Ptd-smad7 therapeutics

ABSTRACT

The present technology provides methods and compositions for the treatment of inflammatory and/or tissue damage conditions. In particular, the use of Smad7 compositions delivered locally or systemically to a site of inflammation and/or tissue damage is described. Other specific embodiments concern treatment or prevention of side effects caused by radiation and/or chemotherapy, including but not limited to oral and gastric mucositis. Also provided are codon-optimized nucleic acids encoding for Smad7 fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage Application under 35 U.S.C.§371 of International Application Ser. No. PCT/US2014/022052, filed onMar. 7, 2014, which claims the benefit of and priority to U.S.provisional patent application U.S. Ser. No. 61/775,252, filed Mar. 8,2013, both of which are incorporated herein by reference in theirentireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under grant numberAR061796 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 29, 2014, isnamed 089491-0303_SL.txt and is 120,505 bytes in size.

BACKGROUND

Oral mucositis, a severe oral ulceration, is a common adverse effect ofa large dose of radiation for bone marrow transplant or craniofacialradiotherapy for cancer. Severe oral mucositis could require feedingtubes, management of severe pain, and prematurely halting radiotherapy.Excessive inflammation and epithelial ablation are key features of oralmucositis.

Palifermin, a KGF (human keratinocyte growth factor) recombinantprotein, is approved for preventing oral mucositis in bone-marrowtransplant patients. Two Palifermin clinical trials in head and neckcancer patients showed that Palifermin reduced severe oral mucositisincidence from 67% and 69% to 51% and 54%, respectively. Other oralmucositis drugs in clinical trials or pre-clinical studies includegrowth factors, agents for radioprotection, anti-inflammatory agents orimmune modulators.

The modest effects of Palifermin and drugs being developed in the abovementioned categories highlight the need for identification of biomarkersfor novel therapies. However, the lack of routine diagnostic biopsies ordiscarded tissues from oral mucositis patients has hindered this effort.

Cutaneous wound healing progresses through three overlapping phases:inflammation, tissue formation, and tissue remodeling. These are dynamicprocesses that involve interactions among the epidermis, leukocytes,extracellular matrix (ECM), and dermal fibroblasts. In response to skininjury, blood clots, infiltrated inflammatory cells and other cell typesin the wound release multiple cytokines and chemokines. These cytokinesinitiate fibroblast proliferation and synthesis of ECM that fill thewound deficit and lead to wound closure.

Meanwhile, keratinocytes at the wound edge begin to proliferate andmigrate to cover the wound surface. Underneath the re-epithelializedepidermis, new stroma, called granulation tissue, begins to fill thewound space, which contains provisional ECM, inflammatory cells,fibroblasts, and blood vessels. Once the wound area is filled with thegranulation tissue and covered by newly re-epithelialized epidermis, theprocess of wound closure is completed. Later on, the wound graduallyreturns to normal strength and texture through tissue remodeling.

Among the many molecules known to influence wound healing, transforminggrowth factor β (TGF-β) has the broadest spectrum of action, affectingall cell types that are involved in all stages of wound healing (Feng etal., Annu Rev Cell Dev Biol 21:659-693, 2005). The various functions ofTGF-β are mediated by a number of signaling molecules, including theSmad family members. When a ligand binds to TGF-β type I and type IIreceptors (TGFβRI and TGF-βRII), TGF-βRI phosphorylates Smad2 and Smad3.Phosphorylated Smad2 and Smad3 bind a co-Smad, Smad4, to formheteromeric Smad complexes and translocate into the nucleus to regulatetranscription of TGF-β target genes.

TGF-β signaling has been reported to exert both positive and negativeeffects on wound healing (Wang et al., J Investig Dermatol Symp Proc 11:112-117, 2006). For instance, Smad3 deficient mice, in which TGF-βsignaling is partially abrogated, exhibit accelerated wound healing(Ashcroft et al., Nat Cell Biol 1:260-266, 1999). In contrast, theintroduction of exogenous Smad3 to wound sites to enhance TGF-βsignaling also accelerated wound healing in a rabbit dermal ulcer model(Sumiyoshi et al., J Invest Dermatol 123:229-236, 2004). Skin wounds inSmad4-deficient mice have a dramatic increase in inflammation andangiogenesis causing a delay in wound closure and formed an excessivescar (Owens et al., Am J Pathol 176:122-133, 2010). Transient adenoviralgene transfer of Smad7, an antagonist of TGF-β signaling, in cornealepithelium and stroma resulted in accelerated corneal wound healing withreduced inflammation (Saika et al., Am J Pathol 166:1405-1418, 2005).Further, Smad7 gene transfer to the lens epithelium and stroma preventedinjury-induced epithelial-mesenchymal transition of lens epithelialcells and suggests a potential role of Smad7 in prevention of capsularfibrosis (Saika et al., Lab Invest 84:1259-1270, 2004). However,adenoviral vector delivery of Smad7 to balloon injury in rat carotidarteries resulted in reduced vascular healing (Mallawaarachchi et al.,Arterioscler Thromb Vasc Biol 25: 1383-1387, 2005). These studiessuggest that the effects of TGF-β signaling components, such as Smad7,on wound healing are complex and highly context-specific. Additionally,the effect of Smad7 may not always be explained by its role in TGF-βsignaling. For instance, Smad7 has also been shown to interact withcomponents of the Wnt/β-catenin (Han et al., Dev Cell Biol 11:301-312,2006) and the TNFβ/NF-κB (Hong et al., Nat Immunol 8:504-513, 2007)families.

SUMMARY

The present technology provides a nucleic acid molecule comprising acodon-optimized human Smad7 cDNA nucleotide sequence. In someembodiments, the codon-optimized human Smad7 nucleotide sequence mayinclude one or more codons for arginine optimized for expression in oneor more of bacteria or yeast, including one or more codons for serineoptimized for expression in one or more of bacteria or yeast, and/orincluding one or more codons for histidine optimized for expression inone or more of bacteria or yeast. In some embodiments, thecodon-optimized human Smad7 nucleotide sequence may include 28 serinecodons, 6 histidine codons, and 9 arginine codons optimized forexpression in one or more of bacteria or yeast. In some embodiments, thecodon-optimized human Smad7 nucleotide sequence may be selected from thegroup consisting of SEQ ID NOs: 9, 21, 23, 24, 26, 28, 30, 32-34, 36,38, 39, 87, 89, 91, 93, 96, 97, 99, and 100. In some embodiments, thecodon-optimized human Smad7 nucleotide sequence may have about 65 to 75percent homology to human Smad7 cDNA, may comprise a nucleotide sequenceencoding an N-terminal fragment SMAD7, may comprise a nucleotidesequence encoding a C-terminal fragment of SMAD7, may comprisenucleotides encoding amino acids 2-258 of the human Smad7 protein, maycomprise nucleotides encoding amino acids 259-426 of the human Smad7protein, or may comprise nucleotides encoding amino acids 204-258 of thehuman Smad7 protein. In some embodiments, any of the foregoing mayfurther comprise a nucleotide sequence encoding a protein transductiondomain, such as Tat. In some embodiments, any of the foregoing may alsofurther comprise a nucleotide sequence encoding one or more of anepitope tag or a purification tag, such as V5,glutathione-S-transferase, or 6-Histidine (SEQ ID NO: 40).

In some embodiments, any of the foregoing may be isolated and/orpurified. In some embodiments, any one of the foregoing may also encodea polypeptide having one or more biological activities selected from thegroup consisting of reducing or eliminating phosphorylation of Smad2,reducing or eliminating nuclear translocation of the NF-κB p50 subunit,increasing cell proliferation, reducing apoptosis, reducingradiation-induced DNA damage, reducing inflammation, reducingangiogenesis, promoting healing in oral mucositis, promoting woundhealing, and treating auto-immune disease. In some embodiments,pharmaceutical compositions comprising the nucleic acid molecules aboveand one or more pharmaceutically acceptable excipients are provided. Insome embodiments, expression vectors comprising the nucleic acidmolecules above operably linked to a promoter are provided, as are hostcells comprising such expression vectors, and pharmaceuticalcompositions comprising such vectors and host cells with one or morepharmaceutically acceptable excipients.

In one aspect, a protein molecule comprising a human Smad7 proteinhaving leucine at position 216 is provided. In some embodiments, thehuman Smad7 protein may be truncated at the C-terminal, or truncated atthe N-terminal. In some embodiments, the truncated human Smad7 proteinmay include about 50% of the full-length Smad7 sequence, or may includeabout 13% of the full-length Smad7 sequence. In some embodiments, thehuman Smad7 protein may comprise or consist of amino acids 2-258, aminoacids 204-258, or amino acids 259-426 of the human Smad7 protein. Insome embodiments, the protein molecule may have one or more biologicalactivities selected from the group consisting of reducing or eliminatingphosphorylation of Smad2, reducing or eliminating nuclear translocationof the NF-κB p50 subunit, increasing cell proliferation, reducingapoptosis, reducing radiation-induced DNA damage, reducing inflammation,reducing angiogenesis, promoting healing in oral mucositis, promotingwound healing, and treating auto-immune disease. In some embodiments,any of the foregoing may further comprise a protein transduction domain,such as Tat. In some embodiments, any of the foregoing may also furthercomprise one or more of an epitope tag or a purification tag, such asV5, glutathione-S-transferase or 6-histidine (SEQ ID NO: 40). In someembodiments, a pharmaceutical composition comprising any of theforegoing, a protein molecule, and one or more pharmaceuticallyacceptable excipients is provided.

In another aspect, a method is provided for treating or preventing aninflammatory condition in a subject comprising providing to the subjecta therapeutically effective amount of the pharmaceutical compositiondescribed above. In some embodiments, the inflammatory condition may beone or more of a chronic wound, skin inflammation, psoriasis, or anautoimmune disease. In some embodiments, the composition may reduceinflammation through inhibition of TGF-β and NF-κB signaling.

In another aspect, a method is provided for preventing or treating adisease or disorder in a subject comprising one or more of increasingone or more of cell proliferation or cell migration, or preventing oneor more of apoptosis or DNA damage in the subject comprising providingto the subject a therapeutically effective amount of the pharmaceuticalcomposition as described above, wherein one or more of increasing one ormore of cell proliferation or cell migration, or preventing one or moreof apoptosis or DNA damage is useful in preventing or treating thedisease or disorder. In some embodiments, the disease or disorder mayinclude one or more of chronic wounds, acute wounds, or mucositis. Insome embodiments, the chronic wounds may include one or more of diabeticulcers, pressure ulcers, venous ulcers, or oral ulcers, the acute woundsmay include one or more of trauma-induced wounds, surgical wounds, orscarring, the mucositis may include one or more of radiation-inducedmucositis or chemotherapy-induced mucositis and the mucositis mayinclude one or more of oral mucositis or gut mucositis.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

Other objects, features and advantages of the present technology willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the presenttechnology, are given by way of illustration only, since various changesand modifications within the spirit and scope of the present technologywill become apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presenttechnology. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-G provide an illustrative embodiment of data showing thatK5.Smad7 mice were resistant to radiation-induced oral mucositis. FIG.1A provides an illustrative embodiment of H&E staining in non-irradiatedand irradiated (day 9 after initiation of radiation) wild-type (WT) andK5.Smad7 tongues. The vertical lines in the images of tongues from WTmice highlight the ulcer boundary and dotted lines in the imagesindicate the epithelial-stromal boundary (scale bar, 50 μm). FIG. 1Bprovides a graphical representation of the quantification of sizes oftongue ulcers (mean±s.e.m); n=8 for WT mice and n=7 for K5.Smad7 mice in8 Gy×3 radiation; n=5 for WT mice and n=4 for K5.Smad7 mice in 18-Gyradiation; n=5 per group for WT and K5.Smad7 mice in 22-Gy radiation.FIG. 1C provides an illustrative embodiment of human ventricularposterior of the tongue (top) and radiation-induced tongue mucositis(bottom) visualized using H&E (left) and CD45 staining (right). Thesolid line indicates the ulcer boundary, and dotted lines indicate thebasement membrane (scale bar, 25 μm). FIG. 1D provides an illustrativeembodiment of immunostaining of CD45, proliferating cell nuclear antigen(PCNA), and TUNEL assay in irradiated sections adjacent to an ulcer fromWT mice and in damaged areas from K5.Smad7 mice (PI, propidium iodide).Dotted lines indicate the basement membrane (scale bar, 25 μm). FIGS.1E-1G provide graphical representations of the quantification ofstaining in FIG. 1D (n=3 or 4 per group). Data are expressed asmean±s.e.m (FIG. 1B) or mean±s.d (FIGS. 1E-1G), and two-tail Studentt-test is used to calculate P values. *P<0.05, **P<0.01, ***P<0.001, NS,no significance determined by two-tailed Student's t-test. Dotted linesin (FIG. 1A), (FIG. 1C) and (FIG. 1D) highlight the basement membrane.Scale bar: 50 μm for all panels in (FIG. 1A) and (FIG. 1C), 25 μm forall panels in (FIG. 1D).

FIGS. 2A-G provide an illustrative embodiment of data showing molecularalterations attenuated by Smad7. FIG. 2A provides an illustrativeembodiment of immune-staining of NF-κB p50, TGF-β1 and pSmad2.Irradiated tongue sections of wild-type (WT) were adjacent to ulcer andsections of K5.Smad7 were from the damaged area. Human samples were fromnon-irradiated oral mucosa and radiation-induced mucositis. Dotted linesdelineate epithelial-stromal boundary. Scale bar, 25 μm for all panels.FIG. 2B provides a graphical representation of the quantification ofimmunostaining of NF-κB p50 and pSmad2 shown in (FIG. 2A). FIG. 2Cprovides an illustrative embodiment of qRT-PCR of TGF-β1 (normalized byKeratin 5, n=6 per group for day 0, n=4 for day 7 and day 9, and n=7 forday 10). FIG. 2D provides a graphical representation of thequantification of human oral keratinocyte migration (see images in FIG.8). Scrambled, scrambled siRNA; n=3 per group. FIG. 2E provides anillustrative embodiment of a western analysis of knockdown efficiency ofsiSmad7-1 and siSmad7-2 for Smad7 and for Rac 1, 72 hours after Smad7knockdown. M, molecular markers. FIG. 2F provides an illustrativeembodiment of western analysis of total and activated (GTP-bound) Rac1protein. M: molecular markers. FIG. 2G provides a graphicalrepresentation of the quantification of the effect of Rac1 knockdown onSmad7-mediated keratinocyte migration (see knockdown efficiency in FIG.9A and images in FIG. 9D). n=3 per group. Data are presented asmean±s.d. and two-tail Student's t-test was used to calculate P valuesfor (FIG. 2B-2D) and (FIG. 2G). *P<0.05, **P<0.01, ***P<0.001. NS, nosignificance.

FIGS. 3A-H provide an illustrative embodiment of data showing Smad7increased Rac1 expression by repressing individual Smad and CtBP1binding to the SBE of the Rac1 promoter. FIG. 3A provides a graphicalrepresentation of the quantification of Rac1 mRNA in wild-type (WT) andSmad7 transgenic keratinocytes. n=4 per group. FIG. 3B provides anillustrative embodiment of western analysis of GTP-Rac1 and total Rac1in WT and Smad7 keratinocytes. Smad7 protein levels in WT and Smad7keratinocytes were determined by reprobing the tubulin western blot withan antibody to Smad7 (see an additional western blot and quantificationin FIGS. 10A-B). FIG. 3C provides an illustrative embodiment of westernanalysis of Rac1 protein level after knocking down individual Smad2,Smad3 or Smad4 in human keratinocytes (see FIG. 10C-10E for Smadknockdown efficiencies). FIG. 3D provides an illustrative embodiment ofa ChIP assay for Smad-2, -3, -4, and -7 binding to the -1.5 Kb SBE siteof the Rac1 promoter in WT and Smad7 transgenic keratinocytes. FIG. 3Eprovides a graphical representation of the quantification of Rac1luciferase reporter assay in mouse keratinocytes. Scrambled: scrambledsiRNA. n=6. FIG. 3F provides a graphical representation of thequantification of the activities of Rac1 luciferase reporter containingSBE or mutant (mut) SBE in WT or Smad7 transgenic keratinocytes. n=6.FIG. 3G provides an illustrative embodiment of images of ChIP assays ofCtBP1 binding to the SBE-1.5 Kb site of the Rac1 promoter in WT orK5.Smad7 keratinocytes. FIG. 3H provides a graphical representation ofChIP-qPCR quantification of CtBP1 binding to the SBE shown in FIG. 3G inWT and Smad7 transgenic keratinocytes. n=4. Data are presented asmean±s.d. and two-tail Student's t-test is used to calculate P valuesfor FIGS. 3A, 3E, 3F and 3H. *P<0.05, **P<0.01, ***P<0.001.

FIGS. 4A-G provide an illustrative embodiment of data showingCtBP1-associated Rac1 repression contributed to inhibition ofkeratinocyte migration. FIG. 4A provides an illustrative embodiment ofwestern analysis of Rac1 protein after knockdown of CtBP1 in human oralkeratinocytes. FIG. 4B provides a graphical representation of thequantification of SBE-containing Rac1 luc reporter activity. n=6. FIG.4C provides a graphical representation of the quantification of theeffect of CtBP 1 knockdown on human oral keratinocyte migration. n=3 pergroup. FIG. 4D provides an illustrative embodiment of immunostaining ofCtBP1. Irradiated sections were adjacent to the ulcer (WT) or thedamaged area (K5.Smad7). Dotted lines denote the basement membrane.Scale bar, 50 μm for all panels. FIG. 4E provides an illustrativeembodiment of immunostaining of CtBP 1 in non-irradiated oral mucosa andradiation-induced oral mucositis in human specimens. Dotted lines denotethe basement membrane. Scale bar, 50 μm for both panels. FIG. 4Fprovides a graphical representation of the quantification of CtBP1nuclear positive cells in FIGS. 4D-E. n=3 or 4 per group. FIG. 4Gprovides a graphical representation of the quantification of qRT-PCR forCtBP1 (normalized with Keratin K5). n=6 per group for day 0, n=4 for day7 and day 9, and n=7 for day 10. Data are presented as mean±s.d. andtwo-tail Student's t-test is used to calculate P values for FIGS. 4B,4C, 4F and 4G. *P<0.05, **P<0.01, ***P<0.001.

FIGS. 5A-G provide an illustrative embodiment of data showing oralTat-Smad7 application prevented radiation-induced oral mucositis inmice. FIG. 5A provides a graphical representation of the quantificationof oral mucositis ulcer sizes on day 9 after initiation of 8 Gy×3radiation. Vehicle=saline or 50% glycerol/PBS. FIG. 5B provides anillustrative embodiment of pathological alterations on day 9 ofinitiation of 8 Gy×3 radiation. Vehicle=saline or 50% glycerol/PBS.Scale bar, 50 μm for H&E panels and 25 μm for remaining panels. Dottedlines delineate epithelial-stromal boundary; the solid line highlightsthe ulcer boundary. FIGS. 5C, 5D, 5E, 5F, and 5G provide a graphicalrepresentation of the quantification of immunostaining shown in FIG. 5B.n=3 or 4 per group. Data are presented as mean±s.e.m (FIG. 5A) ormean±s.d. (FIGS. 5C-5G) and two-tail Student's t-test is used tocalculate P values. *P<0.05, **P<0.01, ***P<0.001. NS, no significance.

FIGS. 6A-G provide an illustrative embodiment of data showing Tat-Smad7treatment on oral mucositis. FIG. 6A provides a graphical representationof the quantification of ulcer sizes measured on day 10 after initiationof 8 Gy×3 radiation. Glycerol=50% glycerol/PBS. FIG. 6B provides anillustrative embodiment of H&E staining of oral mucosa. Upper panels:open ulcer in Palifermin treated but not Tat-Smad7 treated mucosa. Lowerpanels: comparison of epithelial thickness between Palifermin treatedand Tat-Smad7 treated mucosa. Dotted lines delineate the basementmembrane. The vertical line highlights the ulcer boundary. Scale bar, 50μm for all panels. FIG. 6C provides an illustrative embodiment ofimmune-staining of Tat-Smad7 treatment in 20 Gy-induced oral mucositisafter ulcers healed. V5 immunostaining visualizes Tat-Smad7 in oralepithelia (sections are away from the damaged regions). K14immunostaining was used as counterstain. Dotted lines delineate basementmembrane. Scale bar, 25 μm for all panels. FIG. 6D provides anillustrative embodiment of Rac1 western analysis of Tat-Smad7 treatedmouse tongues, day 10 after initiation of 8 Gy×3 radiation. FIG. 6Eprovides an illustrative embodiment of Rac1 western analysis onTat-Smad7 treated normal human oral keratinocytes 48 hours aftertreatment. FIG. 6F provides an illustrative embodiment of the effect ofTat-Smad7 treatment on oral human keratinocyte migration (NOK-SI, seeimages in FIG. 13A). n=4 per group. FIG. 6G provides a graphicalrepresentation of the quantification of survival curves of NOK-SIkeratinocytes and SCC lines (Ca127 and MSK921) with or without Tat-Smad7treatment. n=4 per group for each radiation dose. Data are presented asmean±s.e.m (FIG. 6A) or mean±s.d. (FIGS. 6F, 6G) and two-tail Student'st-test is used to calculate P values. *P<0.05, **P<0.01, ***P<0.001. NS,no significance.

FIGS. 7A-E provide an illustrative embodiment of data showing K5.Smad7oral mucosal tissues were resistant to radiation-induced oral mucositis.FIG. 7A provides an illustrative embodiment of Smad7 western blots:undetectable in non-irradiated wild-type (WT) tongue and barelydetectable after radiation. K5.Smad7 tongues have comparable Smad7protein levels before and after radiation. M: molecular marker. FIG. 7Bprovides an illustrative embodiment of Smad7 immunostaining. Note thatnuclei in some irradiated epithelial cells are hypertrophic. Dottedlines delineate epithelial-stromal boundary. FIG. 7C provides agraphical representation of the quantification of reduced incidence oforal mucositis-induced morbidity in K5.Smad7 mice. Fisher's exact testis used to calculate the p value. **P=0.007. FIG. 7D provides anillustrative embodiment of immune-staining of K5.Smad7 tongue showingreduced infiltration of neutrophils (Ly-6G), macrophages (BM8) andactivated T cells (CD4) compared to WT oral mucositis. Dotted linesdelineate epithelial-stromal boundary. FIG. 7E provides an illustrativeembodiment of immune-staining showing no significant difference inpSmad1/5/8-nuclear positive cells (green) between WT and K5.Smad7 oralmucosa before or after radiation. Keratin (K14) immunostaining (red)highlights the epithelial compartment. Note that nuclei of irradiatedepithelial cells are hypertrophic. The scale bar is 50 μm for allpanels.

FIGS. 8A-D provide an illustrative embodiment of data showing migrationin spontaneously immortalized human oral epithelial cells (NOK-SI) wasdelayed by knocking down Smad7 but accelerated by knocking down TGF-β1.FIGS. 8A and 8B provide an illustrative embodiment of representativeimages of cell migration. Pairs of dotted lines delineate the scratchwound. Quantification of cell migration and efficiency of Smad7knockdown are presented in FIG. 2D and FIG. 2E (above). Scrambled,scrambled siRNA. FIG. 8C provides a graphical representation of thequantification of cell migration after TGF-β1 knockdown from 3 separateexperiments. FIG. 8D provides a graphical representation of qRT-PCRshowing TGF-β1 knockdown efficiency. Data are presented as mean±s.d. andtwo-tail Student's t-test was used to calculate P values. *P<0.05,**P<0.01. NS, no significance.

FIGS. 9A-D provide an illustrative embodiment of data showing knockingdown Rac1 reduced proliferation and migration of wild-type (WT) andSmad7 transgenic keratinocytes. FIG. 9A provides an illustrativeembodiment of western blot analysis for Rac1 48 hours after Rac1 siRNA(siRac1-1, siRac1-2) transfection. Control, scrambled siRNA. FIG. 9Bprovides a graphical representation of the percentage of BrdU labeledcells in WT and Smad7 cultured cells in BrdU incorporation assay with orwithout Rac1 knockdown. Data from 3 separate experiments were presentedas mean±s.d. ***P<0.001. FIG. 9C provides an illustrative embodiment ofrepresentative immunofluorescence of BrdU positive cells presented in(FIG. 9B). An antibody against keratin 14 (K14, red) was used forcounterstain. FIG. 9D provides an illustrative embodiment of in vitrocell migration assay for Smad7 transgenic and WT keratinocytes afterRac1 knockdown. Pairs of dotted lines delineate the scratch wound.Quantification of cell migration is presented in FIG. 2G.

FIGS. 10A-F provide an illustrative embodiment of data showing Smad7increased Rac1 expression by repressing Smad and CtBP1 binding to theSBE of the Rac1 promoter. FIG. 10A provides an illustrative embodimentof western blot analysis for GTP-Rac1 and total Rac1 in Smad7 transgenickeratinocytes. Additional samples are shown in FIG. 3B. M, molecularmarker. FIG. 10B provides a graphical representation of thequantification of GTP-Rac1, total Rac1 and Smad7 in WT and K5.Smad7keratinocytes shown in FIG. 10A and in FIG. 3B. The protein level in WTkeratinocytes of each blot was normalized as “1”. Data is presented asmean±s.d. and two-tail Student's t-test was used to calculate P values.**P<0.01, ***P<0.001. FIGS. 10C and 10D provide an illustrativeembodiment of western blot analysis for Smad2, Smad3, and Smad4knockdown in NOK-SI cells. Their effects on Rac1 expression are shown inFIG. 3C. M, molecular marker. GAPDH, internal protein control byreprobing same blot. FIG. 10F provides an illustrative embodimentshowing CtBP 1 knockdown promotes NOK-SI cell migration. Pairs of dottedlines delineate the scratch wound. Quantification of cell migration andefficiency of CtBP1 knockdown are shown in FIG. 4A and FIG. 4C.

FIGS. 11A-G provide an illustrative embodiment of data showing thepurification and characterization of Tat-Smad7 and Tat-Cre proteins.FIG. 11A shows an illustrative embodiment of a schematic representationof Tat-Smad7 protein. FIG. 11A discloses SEQ ID NOs: 49 and 101,respectively, in order of appearance. FIG. 11B provides an illustrativeembodiment of a western blot of purified Tat-Smad7 protein. FIG. 11Cprovides an illustrative embodiment of immune-staining of Tat-Smad7protein transduction in keratinocytes. Left and middle panels: Tat-Smad7staining (green) using a V5 antibody, counterstained with a K14 antibody(red). Cells showed Tat-Smad7 in the nucleus 5 min after transductionand in both nucleus and cytoplasm 12 hours after transduction. Rightpanels: Tat-Smad7 abrogated Smad2 phosphorylation (pSmad2, green). V5(red) counterstain visualizes Tat-Smad7 transduced cells. FIG. 11Dprovides an illustrative embodiment of immune-staining showing that V5antibody staining detects Tat-Smad7 transduction in buccal mucosa 12hours after Tat-Smad7 topical application. A K14 antibody was used forcounterstain. Scale bar, 50 μm for both panels. FIG. 11E provides anillustrative embodiment of a western blot of purified Tat-Cre proteinwith the same Tat and V5 tags shown in FIG. 11A. FIG. 11F provides anillustrative embodiment of an agarose gel showing activity of Tat-Cre:Tat-Cre cuts out a 1,460 bp floxed fragment from the 7,650 bp vectorpLL3.7. FIG. 11G provides a graphical representation showing Tat-Smad7protein preventive treatment reduced 20 Gy radiation-induced oralulcers. Data are expressed as mean±s.e.m. Two-tail Student's t-test isused to calculate P values. *P<0.05, ***P<0.001.

FIGS. 12A-I provide an illustrative embodiment of data showing effectsof Tat-Smad7 treatment on oral mucositis. FIG. 12A provides a graphicalrepresentation of the quantification of reduced ulcer size in Tat-Smad7(0.8 μg daily, day 6 to day 9) treated oral mucosa. Samples wereharvested on day 10. n=8 per group. FIG. 12B provides an illustrativeembodiment of immunostaining of molecular markers for samples from FIG.12A. Scale bar, 50 μm for the top two panels and 25 μm for other panels.Propidium iodide (PI) and K14 were used as counterstain. FIGS. 12C-Gprovide graphical representation of the quantifications ofimmunostaining shown in FIG. 12C. 3-4 samples were used. FIG. 12Hprovides a graphical representation quantification of the Luciferaseassay. Tat-Smad7 treatment increased the activity of the Rac1 promoterwith SBE but not the mutant SBE in mouse keratinocytes. FIG. 12Iprovides an illustrative embodiment of a ChIP assay for CtBP1 binding tothe SBE of mouse Rac1 promoter in Tat-Smad7 treated mouse keratinocytes.Data are expressed as mean±s.e.m (a) or mean±s.d (c-h) and two-tailStudent's t-test is used to calculate P values. *P<0.05, **P<0.01,***P<0.001. NS, no significance.

FIGS. 13A-H provide an illustrative embodiment of data showing effectsof Tat-Smad7 treatment on migration of human keratinocytes and tumorcell lines. FIG. 13A provides an illustrative embodiment showingTat-Smad7 accelerates NOK-SI cell migration. Quantification from fourseparate experiments is shown in FIG. 6F (above). Pairs of dotted linesdelineate initial wounds. FIG. 13B provides an illustrative embodimentof immunostaining of Tat-Smad7 treatment in NOK-SI cells showingattenuated radiation-induced pSmad2 and NF-κB p50 nuclear localization.FIG. 13C provides an illustrative embodiment showing V5 staining ofMSK921 cells 2 hours after Tat-Smad7 treatment. K14 staining was used ascounterstain. FIG. 13D provides an illustrative embodiment of a Rac1western analysis in MSK921 60 hours after Tat-Smad7 treatment. M,molecular marker. FIG. 13E provides a graphical representation ofquantification of MSK921 cell migration from 3 separate experiments.FIG. 13F provides an illustrative embodiment showing a representativeMSK921 cell migration assay treated with Tat-Smad7 and PBS. Pairs ofsolid lines delineate initial wounds. Dotted lines highlight theforefront of migrated cells. FIG. 13G provides a graphicalrepresentation of quantification of Ca127 cell migration from 3 separateexperiments. FIG. 13H provides an illustrative embodiment showingrepresentative images for FIG. 13G. Pairs of solid lines delineateinitial wounds. Dotted lines highlight the forefront of migrated cells.Data are expressed as mean±s.d. and the two-tail Student's t-test isused to calculate P values. NS, no significance.

FIGS. 14A-B show an illustrative schematic of a summary of potentialmechanisms of Smad7-mediated protection and healing of oral mucositis.FIG. 14A shows an illustrative schematic of how radiation activatesNF-κB, increases TGF-β1 and CtBP1. NF-κB and TGF-β1 induce inflammation.TGF-β1 induces apoptosis, growth arrest and activates Smad-2, -3 and -4,which recruit CtBP1 to the Rac1 promoter to repress Rac1 transcription,leading to blunted re-epithelialization. FIG. 14B shows an illustrativeschematic of how Smad7 blocks NF-κB and TGF-β1-induced inflammation andblocks TGF-β1-induced apoptosis and growth arrest. Smad7 relieves Rac1transcriptional repression by either preventing TGF-β1-mediated Smadactivation (phosphorylation) or competing with signaling Smads/CtBP1transcriptional repression complex in binding to the Rac1 promoter.Increased Rac1 induced by Smad7 contributes to keratinocyte migrationduring re-epithelialization.

FIG. 15 shows an illustrative schematic of Smad 7 domains associatedwith protein partners, potential target effects, and potentialphysiological effects.

FIGS. 16A-B are graphs demonstrating the ability of truncated Smad7proteins to accelerate wound healing in a mouse wound healing model.FIG. 16A is a graph showing the effect of C-terminally truncated(259-426aa) Tat-C-Smad7 on average percent wound healing over timerelative to full-length Tat-Smad7 and control (PBS). n=3 for each group.FIG. 16B is a graph illustrating the effect of Tat-N-Smad7 (1-258aa) onaverage percent wound healing over time relative to full-lengthTat-Smad7 and control (PBS). n=6 for each group. Data are presented asmean±s.d., and two-tail Student's t-test was used to calculate P values.*p<0.05 compared to control (PBS), #p<0.05 compared to Tat-Smad7.

FIGS. 17A-C are photographs and graphs demonstrating that Smad7accelerates wound healing in an impaired wound-healing model. FIG. 17Ais a digital photograph illustrating the gross appearance of wounds indiabetic (db/db) mice treated with PBS or Tat-Smad7 over a time periodof thirteen days. FIG. 17B is a graph showing the effect of Tat-Smad7 onaverage percent wound healing over time relative to REGRANEX® andcontrol (PBS). n=6 for each group. Data are presented as mean±s.d., andtwo-tail Student's t-test was used to calculate P values. *p<0.05compared to control (PBS). FIG. 17C is a histological comparison ofwound samples taken eight days after wounding. The vertical dotted linesin the image from the control (PBS) db/db mouse (top panel) highlightthe wound boundary.

DETAILED DESCRIPTION

As further described herein, the disclosure provides Smad7 proteins andbiologically active fragments and derivatives thereof, nucleic acidsencoding such proteins, vectors including such nucleic acids, and cellsencompassing the vectors, nucleic acids, and/or proteins all for use informulating medicaments and for treating and/or preventing one or morediseases or disorders. Also provided are methods for making and forscreening Smad7 proteins and biologically active fragments andderivatives thereof useful for treating and/or preventing one or morediseases or disorders. Also provided are methods for predicting and/orevaluating a response to treatment using one or more markers associatedwith exposure to Smad7. Such markers may include, but are not limitedto, Rac1 for cell migration, NF-κB for inflammation, and TGF-β forgrowth arrest and inflammation.

Smad7 treatable diseases and disorders may include those including oneor more of reduced cell proliferation, reduced cell migration, increasedcell death, excessive inflammation, and/or DNA damage. Smad7 treatablediseases and disorders may include those where treatment with a Smad7protein and biologically active fragments and derivatives thereof thathave one or more activities including but not limited to increasingproliferation, reducing or inhibiting cell death, reducing excessiveinflammation, preventing DNA damage, and/or increasing cell migration.Such diseases and/or disorders may include but are not limited to acute(e.g., through surgery, combat, trauma) and chronic wounds (e.g.,ulcers, such as diabetic, pressure, venous), scarring, fibrosis, andaberrant healing, mucositis (e.g., oral and/or gastro-intestinal),stomatitis, proctitis, autoimmune disease (e.g., psoriasis, arthritis),and cancer.

It is critical for oral mucositis prevention and treatment to overcomeepithelial ablation due to massive apoptosis and blunted keratinocyteproliferation. The proliferative and anti-apoptotic effects of Smad7 aremore obvious in oral mucositis than in normal oral mucosa, when TGF-β1,a potent growth inhibitor and apoptosis inducer for epithelial cells,was increased.

Although not wishing to be bound by theory, it is believed thatincreased Rac1 activation is largely responsible for Smad7-mediatedkeratinocyte migration in wound closure. This finding was unexpected,given the documented role of TGF-β signaling in Rho/Rac activation incancer cells via a Smad-independent mechanism (Dernyck et al., Nature415:577-584, 2003).

It is believed that during oral mucositis, Smad-dependent Rac1repression overcomes Smad-independent Rac1 activation (if any) due toincreased Smad signaling (evidenced by increased pSmad2) and Smadtranscriptional co-repressor CtBP1. When this repression is abrogated bySmad7, it permits Rac1 activation-mediated keratinocyte migration.However, in oral cancer cells, signaling Smads are lost or inactivated,or other mechanisms independently activate Rac1. As a result,Smad7-mediated abrogation of Rac1 repression would no longer occur.

Although Rac1 activation also contributed to keratinocyte proliferation,knocking down Rac1 only partially attenuated the proliferative effect ofSmad7. Therefore, Rac1's contribution to proliferation appears to belimited, and blocking TGF-β1-induced growth arrest is also needed toovercome radiation-induced growth inhibitory effects.

Dampening excessive inflammation creates a microenvironment for oralmucositis healing. The antagonistic effect of Smad7 on both TGF-β andNF-κB signaling makes Smad7 a more efficient anti-inflammatory moleculethan other agents targeting only NF-κB. Because inflammatory cellsproduce cytokines that further activate TGF-β and NF-κB, reduced TGF-βand NF-κB signaling, found in K5.Smad7 or Tat-Smad7 treated oral mucosaafter radiation, reflects the direct antagonistic effect of Smad7 onthese two pathways and the consequence of reduced inflammatory cytokinesfrom infiltrated leukocytes. However, Smad7 did not reduce NF-κB orTGF-β signaling below their normal physiological conditions. Thisincomplete blockade of NF-κB or TGF-3 signaling may be beneficial tooral mucositis healing, as a complete loss of either pathway couldinduce excessive inflammation.

The primary obstacle to using growth factors to treat oral mucositis incancer patients is the potential risk of promoting cancer cell growth.The majority of human oral cancers lose TGF-3 signaling in tumorepithelial cells. Thus, anti-Smad-associated cell proliferation andmigration by Smad7 would not be effective in cancer cells. In tumorswith intact TGF-β signaling, activation of other oncogenic pathwayscould override TGF-3-induced tumor suppressive effects. These twoscenarios could explain why there was no observation of Smad7 increasingproliferation and migration in oral cancer cells with mutant or intactTGF-β signaling components.

Additionally, TGF-β signaling promotes tumor invasion mainly throughSmad-independent mechanisms after loss of TGF-β-induced tumorsuppression. Thus, blocking TGF-β signaling by Smad7 in cancer cellscould abrogate TGF-β-mediated tumor promotion effects, which behavessimilarly to TGF-3 inhibitors currently being used in clinical trialsfor advanced cancers. Further, the potent anti-inflammatory effect ofSmad7 may reduce the risk of tumor progression. Therefore, long-termSmad7 application may also be helpful in cancer treatment.

Spontaneous tumor formation in K5.Smad7 mice has not been observed.Because Smad7 is not a secreted protein, local and short-term Smad7protein delivery in oral mucositis treatment should have few systemiceffects. In bone marrow transplant patients, whose oral epithelia do notcontain cancer cells, Smad7 topical application may be suitable for bothprevention and treatment of oral mucositis.

Although not wishing to be bound by any theory, Smad7-mediated oralmucositis healing appears to be a result of targeting multiplepathogenic processes mediated by one or more molecules (see, e.g., FIGS.14A-B). It is believed that one or more of these molecules (e.g., TGF-β,NF-κB, CtBP1, Rac1) may also be helpful as predictive and therapeuticresponsive markers of oral mucositis in patients.

A. Nucleic Acids, Vectors and Host Cells

The present disclosure also provides, in another embodiment, genesencoding Smad7. In addition to the wild-type SMAD7 gene (SEQ ID NOs: 22,88), as well as various codon-optimized versions (SEQ ID NOs: 9, 21, 23,24, 26, 28, 30, 32-34, 36, 38, 39, 87, 89, 91, 93, 96, 97, 99, and 100),it should be clear that the present technology is not limited to thespecific nucleic acids disclosed herein. As discussed below, a “Smad7gene” may contain a variety of different bases and yet still produce acorresponding polypeptide that is functionally indistinguishable from,and in some cases structurally identical to, the human gene disclosedherein.

1. Nucleic Acids Encoding Smad7

Nucleic acids according to the present technology may represent anentire Smad7 gene, a truncated portion, and/or a fragment of Smad7 thatexpresses a polypeptide with one or more activity associated with Smad7such as but not limited to increasing proliferation, reducing orinhibiting cell death, reducing excessive inflammation, preventing DNAdamage, and/or increasing cell migration, as well as treating orpreventing one or more disease or disorders in which such treatmentwould be helpful as further discussed herein. Such activities can beassessed using one or more assays including, but not limited to, theability to block phosphorylation of Smad2 and/or nuclear translocationof the NF-κB p50 subunit, increase cell proliferation, reduce apoptosisand/or radiation-induced DNA damage, reduce inflammation and/orangiogenesis, promote healing in oral mucositis, surgical wounds,diabetes wounds, and/or wounds associated with chronic inflammation inmice. The nucleic acid may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In particularembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also provided is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present technology may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

As used in this application, the term “a nucleic acid encoding a Smad7”may refer to a nucleic acid molecule that has been isolated free oftotal cellular nucleic acid and/or may refer to a cDNA encoding a Smad7polypeptide. As used herein, the term “isolated free of total cellularnucleic acid” means that the nucleic acid molecule is about or at leastabout 75% pure, 80% pure, 85% pure, 90% pure, 95% pure, 96% pure, 97%pure, 98% pure, 99% pure, or 100% pure of other cellular nucleic acidmolecules as determined using standard biochemical techniques, such asbut not limited to agarose gel electrophoresis. As used herein, the term“isolated free of total cellular protein” means that the proteinmolecule is about or at least about 75% pure, 80% pure, 85% pure, 90%pure, 95% pure, 96% pure, 97% pure, 98% pure, 99% pure, or 100% pure ofother cellular nucleic acid molecules as determined using standardbiochemical techniques, such as but not limited to a western blot. Incertain embodiments, the present technology concerns a nucleic acidsequence essentially as set forth in, and/or including any one of SEQ IDNOs: 9, 21, 23, 24, 26, 28, 30, 32-34, 36, 38, 39, 87, 89, 91, 93, 96,97, 99, and 100.

An isolated nucleic acid molecule may be produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis. Isolated nucleic acid molecules includenatural nucleic acid molecules and homologues thereof, including, butnot limited to, natural allelic variants and modified nucleic acidmolecules in which nucleotides have been inserted, deleted, substituted,and/or inverted in such a manner that such modifications provide thedesired effect (e.g., production of Smad7 protein in non-humanexpression systems).

The term “essentially as set forth in one or more nucleic acid sequence(e.g., SEQ ID NOs: 9-11, 21, 23-41” means that the nucleic acid sequencesubstantially corresponds to at least a portion, and in some cases theentirety, of the one or more nucleic acid sequence (e.g., SEQ ID NOs: 9,21, 23, 24, 26, 28, 30, 32-34, 36, 38, 39, 87, 89, 91, 93, 96, 97, 99,and 100). In some embodiments, sequences that substantially correspondto at least a portion of a nucleic acid sequence, may correspond toabout, or at least about 50 nucleic acids, 75 nucleic acids, 150 nucleicacids, 200 nucleic acids, 250 nucleic acids, 300 nucleic acids, 350nucleic acids, 400 nucleic acids, 450 nucleic acids, 500 nucleic acids,550 nucleic acids, 600 nucleic acids, 650 nucleic acids, 700 nucleicacids, 750 nucleic acids, 800 nucleic acids, 900 nucleic acids, 1000nucleic acids, 1100 nucleic acids, 1200 nucleic acids, or 1250 nucleicacids of one or more of the sequences described herein. In someembodiments, sequences that substantially correspond to at least aportion of a nucleic acid sequence, may correspond to about a range ofabout 50-1250 nucleic acids, 75-1250 nucleic acids, 150-1250 nucleicacids, 200-1250 nucleic acids, 250-1250 nucleic acids, 300-1250 nucleicacids, 350-1250 nucleic acids, 400-1250 nucleic acids, 450-1250 nucleicacids, 500-1250 nucleic acids, 550-1250 nucleic acids, 600-1250 nucleicacids, 650-1250 nucleic acids, 700-1250 nucleic acids, 750-1250 nucleicacids, 800-1250 nucleic acids, 900-1250 nucleic acids, 1000-1250 nucleicacids, 1100-1250 nucleic acids, 1200-1250 nucleic acids, at least about50-75 nucleic acids, 75-150 nucleic acids, 75-200 nucleic acids, 75-250nucleic acids, 75-300 nucleic acids, 75-350 nucleic acids, 75-400nucleic acids, 75-450 nucleic acids, 75-500 nucleic acids, 75-550nucleic acids, 75-600 nucleic acids, 75-650 nucleic acids, 75-700nucleic acids, 75-750 nucleic acids, 75-800 nucleic acids, 75-900nucleic acids, 75-1000 nucleic acids, 75-1100 nucleic acids, 75-1200nucleic acids, or 75-1250 nucleic acids or 1250 nucleic acids of one ormore of the sequences described herein.

In some embodiments, sequences that substantially correspond to at leasta portion of a nucleic acid sequence include identical sequences to thatportion of the nucleic acid sequence. In some embodiments, sequencesthat substantially correspond to at least a portion of a nucleic acidsequence or the entirety of a nucleic acid sequence may include one ormore functionally equivalent codons. The term “functionally equivalentcodon” is used herein to refer to one or more codons that encode thesame amino acid, such as the six codons for arginine or serine, and insome embodiments refers to codons that encode biologically equivalentamino acids, as discussed in the following pages. The term “biologicallyequivalent” amino acid is used herein to refer to one or more aminoacids that when changed from the amino acid present in the amino acidsequence of human Smad7 wild-type protein, do not change one or more (orin some embodiments any) of the biological activities of Smad7 describedherein, such as but not limited to, increasing proliferation, reducingor inhibiting cell death, reducing excessive inflammation, preventingDNA damage, and/or increasing cell migration, as well as treating orpreventing one or more disease or disorders in which such treatmentwould be helpful as further discussed herein.

In some embodiments, allowing for the degeneracy of the genetic code,sequences that have about or at least about 60%, 70%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% of nucleotides that areidentical to the nucleotides of any one of the codon-optimized nucleicacid sequences (e.g., SEQ ID NOs: 9-11, 21, 23-41) may be consideredsubstantially corresponding nucleic acid sequences. Sequences that areessentially the same as those set forth in any one of the nucleic acidsequences (e.g., SEQ ID NOs: 9-11, 21, 23-41) also may be functionallydefined as sequences that are capable of hybridizing to a nucleic acidsegment containing the complement of SEQ ID NOs: 9-11, 21, 23-41 undervarious standard conditions.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications it is appreciated that lower stringencyconditions are preferred. Under these conditions, hybridization mayoccur even though the sequences of the hybridizing strands are notperfectly complementary, but are mismatched at one or more positions.Conditions may be rendered less stringent by increasing saltconcentration and/or decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Hybridizationconditions can be readily manipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps are introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino acid ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions can then becompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent homology between the two sequences is a function of the numberof identical positions shared by the sequences (% identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments the two sequences are the samelength.

To determine percent homology between two sequences, the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-5877 can be used. Such an algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990) J. Mol Biol.215:403-410. BLAST nucleotide searches is performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a nucleic acid molecules described or disclose herein.BLAST protein searches is performed with the XBLAST program, score=50,wordlength=3. To obtain gapped alignments for comparison purposes,Gapped BLAST may be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (forexample, XBLAST and NBLAST) are used. See the website of the NationalCenter for Biotechnology Information for further details (on the WorldWide Web at ncbi.nlm.nih.gov). Proteins suitable for use in the methodsdescribed herein also includes proteins having between 1 to 15 aminoacid changes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 amino acid substitutions, deletions, or additions, compared tothe amino acid sequence of any protein described herein. In otherembodiments, the altered amino acid sequence is at least 75% identical,for example, 77%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or100% identical to the amino acid sequence of any protein inhibitordescribed herein. Such sequence-variant proteins are suitable for themethods described herein as long as the altered amino acid sequenceretains sufficient biological activity to be functional in thecompositions and methods described herein. In certain instancesconservative amino acid substitutions are utilized. Illustrativeconservative substitution among amino acids are within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine. The BLOSUM62 table is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff et al. (1992), Proc.Natl Acad. Sci. USA, 89:10915-10919). The BLOSUM62 substitutionfrequencies can be used to define conservative amino acid substitutionsthat, in some embodiments, are introduced into the amino acid sequencesdescribed or disclosed herein. Although it is possible to design aminoacid substitutions based solely upon chemical properties (as discussedabove), the language “conservative amino acid substitution” preferablyrefers to a substitution represented by a BLOSUM62 value of greater than−1. For example, an amino acid substitution is conservative if thesubstitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

The DNA segments of the present technology include those encodingbiologically functional equivalent Smad7 proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described elsewhere.

As described in greater detail below, the Smad7 nucleic acid sequencehas been optimized for expression in alternative host organisms (e.g.,non-human). Although as described above, the genetic code is degenerate,so frequently one amino acid may be coded for by two or more nucleotidecodons. Thus, multiple nucleic acid sequences may encode one amino acidsequence. Although this creates identical proteins, the nucleic acidsthemselves are distinct, and can have distinct properties. As describedherein, one aspect of the choice of codon usage can be (but is notlimited to) the ability to express a protein in a non-native cells(e.g., a human protein in bacteria or yeast), or the level of expressionin such cells. In order to obtain enough protein for purification,testing, and use in in vitro assays, in animal models, and eventually inclinical development, efficient protein expression in non-human systemsis needed.

A series of 23 arginine amino acids in the human Smad7 protein sequencecoded for by one or more of AGG (1.7% codon utilization; 9 residues),AGA (2.8% codon utilization; 2 residues), CGA (3.5% codon utilization; 4residues), or CGG (5.4% codon utilization; 8 residues) has beenidentified, and it has been determined that in order to have efficientprotein expression from non-human sources, such as, but not limited to,bacteria and/or yeast that one or more, and potentially all the argininecodons should be modified to CGT (20.6% codon utilization). Therefore,in some embodiments, the Smad7 codon-optimized nucleic acid sequenceincludes at least 1, at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, or 23 codons for arginine that have been changed to CGT. In someembodiments, the Smad7 codon-optimized nucleic acid sequence includesone or more or all of the arginine codons at nucleic acid sequencepositions 7-9, 43-45, 169-171, 403-405, 490-492, 526-528, 526-528,823-825, 1057-1059, 16-18, 136-138, 199-201, 598-600, 31-33, 112-114,316-318, 772-774, 940-942, 973-975, 1135-1137, 1276-1278, 637-639, or814-816 be changed to CGT.

A series of 33 serine residues in the human Smad7 protein sequence codedfor by TCC or TCG (9%) has been identified, and it has been determinedthat it may be beneficial to efficient protein expression andpurification from non-human sources, such as, but not limited to,bacteria and/or yeast, that one or more, and potentially all the serinecodons be modified to AGC (15% codon utilization). Therefore, in someembodiments, the Smad7 codon-optimized nucleic acid sequence includes atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 30, at least 31, at least 32 or 33 codons for serinethat have been changed to (AGC). In some embodiments, the Smad7codon-optimized nucleic acid sequence includes one or more or all of theserine codons at nucleic acid sequence positions 19-21, 46-48, 133-135,292-294, 349-351, 451-453, 454-456, 460-462, 511-513, 514-516, 544-546,595-597, 616-618, 634-636, 691-693, 694-696, 739-741, 745-747, 775-777,847-849, 907-909, 919-921, 943-945, 1006-1008, 1009-1101, 1030-1032,1054-1056, 1093-1095, 1126-1128, 1192-1194, 1237-1239, 1240-1242,1273-1275. Of these, 23 codons (19-21, 292-294, 349-351, 451-453,454-456, 460-462, 511-513, 514-516, 544-546, 616-618, 634-636, 691-693,694-696, 739-741, 745-747, 775-777, 847-849, 907-909, 919-921,1009-1101, 1030-1032, 1054-1056, 1093-1095) can be changed withoutintroducing potential alternative open reading frames.

A series of 12 histidine residues in the human Smad7 protein sequencecoded for by CAC (9.6% codon usage) has also been identified, and it hasbeen determined that it may be beneficial to efficient proteinexpression and purification from non-human sources, such as but notlimited to bacteria and/or yeast, that one or more, and potentially allthe serine codons be modified to CAT (optionally to 12.6% usage).Therefore, in some embodiments, the Smad7 codon-optimized nucleic acidsequence includes at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, or 12 codons for histidine that have been changed to (CAT). Insome embodiments, the Smad7 codon-optimized nucleic acid sequenceincludes one or more or all of the serine codons at nucleic acidsequence positions 142-144, 214-216, 217-219, 220-222, 226-228, 289-291,589-591, 778-780, 1072-1074, 1147-1149. Of these, 4 codons (nucleotides217-219, 220-222, 589-591, 778-780) can be changed without introducingpotential alternative open reading frames.

In some embodiments, one or more codon-optimized nucleic acids mayinclude one or more of at least one and any integer up to 22 of itsarginine codons modified to CGT, at least one and any integer up to 28of its serine codons (optionally that are able to be modified withintroducing open reading frames) modified to AGC, or at least one andany integer up to 12 of its histidine codons (optionally that are ableto be modified with introducing open reading frames) modified to CAT. Insome embodiments, one or more codon-optimized nucleic acid may includeat least one and any integer up to 22 of its arginine codons modified toCGT, at least one and any integer up to 28 of its serine codons(optionally that are able to be modified with introducing open readingframes) modified to AGC, and at least one and any integer up to 12(optionally that are able to be modified with introducing open readingframes) of its histidine codons modified to CAT. In some embodiments,one or more codon-optimized nucleic acid may include 22 of its argininecodons modified to CGT, 28 of its serine codons (optionally that areable to be modified with introducing open reading frames) modified toAGC, and 12 of its histidine codons (optionally that are able to bemodified with introducing open reading frames) modified to CAT. In someembodiments, one or more codon-optimized nucleic acid may also have anucleotide substitution in the codon for Met216 (ATG), to form the codonfor Leu216 (CTG).

In some embodiments, one or more codon-optimized nucleic acids may haveabout 65% to 75%, about 65% to 68%, about 68% to 75%, or about 68% to71% homology to human Smad7 wild-type cDNA (SEQ ID NOs: 22, 88). In someembodiments, one or more codon-optimized nucleic acid may have about65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%, homology tohuman Smad7 wild-type cDNA (SEQ ID NOs: 12, 22). In some embodiments,one or more codon-optimized nucleic acid may also have a nucleotidesubstitution in the codon for Met216 (ATG), to form the codon for Leu216(CTG).

A methionine codon (Met216; ATG) that has the potential for beingperceived by translation machinery (e.g., such as but not limitedbacteria or yeast) as an alternative open reading frame has beenidentified. Although not intending to be bound by theory, it is believedthat the presence of the second potential open reading frame maydecrease expression of the Smad7 protein. In some embodiments, one ormore Smad7 nucleic acid sequences are modified at nucleotide position(646-648) to encode a human Smad7 protein where Met216 (ATG) is modifiedto Leu216 (CTG).

It has also been discovered that various truncated forms and fragmentsof Smad7 protein retain one or more of the activities of full-lengthhuman Smad7, such as, but not limited to, increasing proliferation,reducing or inhibiting cell death, reducing excessive inflammation,preventing DNA damage, and/or increasing cell migration, as well astreating or preventing one or more disease or disorders in which suchtreatment would be helpful as further discussed herein. Such activitiescan be assessed using one or more assays including, but not limited to,the ability to block phosphorylation of Smad2 and/or nucleartranslocation of the NF-κB p50 subunit, increase cell proliferation,reduce apoptosis and/or radiation-induced DNA damage, reduceinflammation and/or angiogenesis, promote healing in oral mucositis,surgical wounds, diabetes wounds, and/or wounds associated with chronicinflammation in mice.

Further, in some embodiments, various truncated forms and fragments ofSmad7 protein retain only a subset of the one or more of the activitiesof full-length human Smad7. For example, the C-terminal MH2 domain ofSmad7 may primarily mediate the anti-inflammatory effect of Smad7. Smad7peptides having this anti-inflammatory function may be sufficient andoptionally an improvement for treating chronic inflammation associatedconditions, such as but not limited to, oral mucositis, stomatitis,arthritis, and psoriasis, among others. The N-terminal MH1 domain mayprimarily mediate cell migration and/or blocking TGF-β-induced growtharrest and/or fibrotic response. Smad7 peptides having this cellmigration and proliferation function may be sufficient, and optionallyan improvement, for enhancing healing that is not associated withexcessive inflammation. Types of wounds that might benefit from thisform of treatment include, but are not limited to, surgical wounds,fibrotic scarring, and diabetes wounds, defective healing and/orscarring among others.

In some embodiments, nucleic acid molecules (optionally codon-optimizednucleic acid molecules as described above and herein) encode fragmentsor truncated forms of Smad7 protein (optionally including Leu216). Insome embodiments, these fragments and/or truncated forms of Smad7protein retain one or more or all of the activities of full-length humanSmad7 protein. In some embodiments, such truncated nucleic acidsequences encode the N-terminal portion of the Smad7 protein. In someembodiments, such truncated nucleic acid sequences encode the C-terminalportion of the Smad7 protein. In some embodiments, such truncatednucleic acid sequences (nucleotide positions 4-774) encode amino acids2-258 of the human Smad7 protein. In some embodiments, such truncatednucleic acid sequences (nucleotide positions 775-1278) encode aminoacids 259-426 of the human Smad7 protein. In some embodiments, suchfragments of the nucleic acid sequences (nucleotide positions 610-774)encode amino acids 204-258 of the human Smad7 protein.

The term “truncated” as used herein in reference to nucleic acidmolecules refers to a molecule that contains nucleotide sequencesencoding the natural N-terminus of a corresponding protein (with orwithout a cleaved leader sequence), but lacks one or more nucleotidesstarting from the C-terminus-encoding portion of the molecule, or amolecule that contains nucleotide sequences encoding the naturalC-terminus of a corresponding protein (with or without a cleaved leadersequence), but lacks one or more nucleotides starting from theN-terminus-encoding portion of the molecule. In some embodiments,molecules lacking nucleotides encoding at least about 25, at least about50, at least about 75, at least about 100, at least about 125, at leastabout 150, at least about 200, at least about 250, at least about 300,or at least about 350, or at least about 400 amino acids from one or theother terminus are specifically provided. Similarly, the term“truncated” may also be used in reference to protein molecules encodedby truncated nucleic acid molecules. In some embodiments, a “truncated”molecule is biologically active, having (or encoding a polypeptidehaving) one or more of the Smad7 activities described herein.

The term “fragment” as used herein in reference to nucleic acidmolecules refers to a molecule containing contiguous residues of a fulllength sequence but lacking some 5′ and/or 3′ sequences of the fulllength sequence. In some embodiments, a “fragment” includes a portion ofone or more of the full length sequences described herein. In someembodiments, the “fragment” does not include sequences encoding eitherthe N-terminal or the C-terminal, but only internal fragments. In someembodiments, a “fragment” encodes a polypeptide that is biologicallyactive, having one or more of the Smad7 activities described herein. Insome embodiments, nucleic acid fragments may encode proteins having atleast about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150 amino acids. Similarly,“fragment” may also be used in reference to protein molecules encoded bySmad7 nucleic acid fragments.

The term “N-terminal portion” as used herein in refers to a fragment ofa corresponding protein that contains the protein's N-terminus but lacksall sequences C-terminal to an internal residue.

The term “C-terminal portion” as used herein in refers to a fragment ofa corresponding protein that contains the protein's C-terminus but lacksall sequences N-terminal to an internal residue.

Although not intending to be bound by theory, the Smad7 protein activityis generally believed to be the result of interactions in both thecytoplasm and nucleus of a cell. For that reason among others, thereexisted a general belief that Smad7 protein was not a candidate for atherapeutic role. However, it was decided to pursue development of Smad7as a protein therapeutic, and modify the Smad7 nucleic acid sequence toencode a protein transduction domain (PTD) in frame with the Smad7nucleic acid sequence (e.g., optionally any nucleic acid sequencedescribed herein encoding Smad7 protein, including human wild-type andcodon-optimized sequences, both full-length and biologically activefragments or truncated portions). In some embodiments, the PTD islocated at the 3′ end of the Smad7 nucleic acid sequence, and in someembodiments the PTD is located at the 5′ end of the Smad7 nucleic acidsequence. In some embodiments, there is a linker sequence encoding 1, 2,3, 4, 5, or 6 amino acids that connects the PTD and the Smad7 nucleicacid sequence.

In some embodiments, the PTD nucleic acid sequence is a Tat nucleic acidsequence. ggccgtaaaaaacgccgtcaacgccgccgt (SEQ ID NO: 1) encodingGRKKRRQRRR (SEQ ID NO: 2), tatggccgtaaaaaacgccgtcaacgccgccgt (SEQ ID NO:3) encoding YGRKKRRQRRR (SEQ ID NO: 4), or ggccgtaaaaaacgccgtcaa (SEQ IDNO: 5) encoding GRKKRRQ (SEQ ID NO: 6).

In some embodiments, the nucleic acid sequence further includes anucleotide sequence encoding one or more of an epitope tag or apurification tag. In some embodiments, the epitope tag is V5. In someembodiments, the purification tag is one or more ofglutathione-S-Transferase (GST) or 6-histidine (H6) (SEQ ID NO: 40).

The term “epitope tag” as used herein in reference to nucleic acidmolecules refers to nucleotides encoding peptide sequences that arerecognized and bound by the variable region of an antibody or fragment.In some embodiments, the epitope tag is not part of the native protein.In some embodiments, the epitope tag is removable. In some embodiments,the epitope tag is not intrinsic to the protein's native biologicalactivity. Examples of epitope tags include, but are not limited to V5.

The term “purification tag” as used herein in reference to nucleic acidmolecules refers to nucleotides encoding peptide sequences thatfacilitate the purification of the protein, but are generally notnecessary for the protein's biological activity. In some embodiments,purification tags may be removed following protein purification.Examples of purification tags include, but are not limited to GST andH-6 (SEQ ID NO: 40).

2. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors are employed to expressthe Smad7 polypeptide product, which can then be purified for varioususes. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell-equipped to construct a vector through standard recombinanttechniques, which are described, e.g., in Sambrook, et al., MolecularCloning (Cold Spring Harbor Lab Press, 1989), and Ausubel, et al.,Current Protocols in Molecular Biology (Wiley, 1994), both incorporatedherein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism, including promoters andenhancers. In addition to control sequences that govern transcriptionand translation, vectors and expression vectors may contain nucleic acidsequences that serve other functions, such as transcription terminationsignals and poly-adenylation sites.

The capacity of certain viral vectors to efficiently infect or entercells, to integrate into a host cell genome and stably express viralgenes, have led to the development and application of a number ofdifferent viral vector systems. Robbins, et al., Pharmacol. Ther.80:35-47 (1998). Viral systems are currently used as vectors for ex vivoand in vivo gene transfer. For example, adenovirus, herpes-simplexvirus, lentiviruses, retrovirus and adeno-associated virus vectors arebeing evaluated currently for treatment of diseases such as cancer,cystic fibrosis, Gaucher disease, renal disease and arthritis. Robbins,et al., Pharmacol. Ther. 80:35-47 (1998); Imai, et al., Nephrologie19:379-402 (1998); U.S. Pat. No. 5,670,488. The various viral vectorspresent specific advantages and disadvantages, depending on theparticular gene-therapeutic application.

Suitable non-viral methods for nucleic acid delivery for transformationof an organelle, a cell, a tissue or an organism for use with thepresent technology are believed to include virtually any method by whicha nucleic acid (e.g., DNA) can be introduced into an organelle, a cell,a tissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference); by calcium phosphate precipitation (Graham, et al., Virology52:456-467 (1973); Chen, et al., Mol. Cell Biol. 7:2745-2752 (1987);Rippe, et al., Mol. Cell Biol. 10:689-695 (1990)); by using DEAE-dextranfollowed by polyethylene glycol (Gopal, Mol. Cell Biol. 5:1188-1190(1985)); by direct sonic loading (Fechheimer, et al., PNAS 84:8463-8467(1987)); by liposome mediated transfection (Nicolau, et al., Biochim.Biophys. Acta 721:185-190 (1982); Fraley, et al., PNAS 76:3348-3352(1979); Nicolau, et al., Methods Enzymol. 149: 157-176 (1987); Wong, etal., Gene 10:87-94 (1980); Kaneda, et al., J. Biol. Chem.264:12126-12129 (1989); Kato, et al., J. Biol. Chem. 266:3361-3364(1991)); by microprojectile bombardment (PCT Application Nos. WO94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055,5,550,318, 5,538,877 and 5,538,880, and each incorporated herein byreference); by agitation with silicon carbide fibers (Kaeppler, et al.,Plant Cell Rep. 9:415-418 (1990); U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); or by PEG-mediatedtransformation of protoplasts (Omirulleh, et al., Plant Mol. Biol.21:415-428 (1993); U.S. Pat. Nos. 4,684,611 and 4,952,500, eachincorporated herein by reference); by desiccation/inhibition-mediatedDNA uptake (Potrykus, et al., Mol. Gen. Genet. 199:169-177 (1985)).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

3. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present technology to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented.

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes (e.g., bacteriaor yeast), depending upon whether the desired result is replication ofthe vector or expression of part or all of the vector-encoded nucleicacid sequences. Numerous cell lines and cultures are available for useas a host cell, and they can be obtained through the American TypeCulture Collection (ATCC), which is an organization that serves as anarchive for living cultures and genetic materials (atcc.org). Anappropriate host can be determined by one of skill in the art based onthe vector backbone and the desired result. A plasmid or cosmid, forexample, can be introduced into a prokaryote host cell for replicationof many vectors. Bacterial cells used as host cells for vectorreplication and/or expression include DH5α, JM109, and KCB, as well as anumber of commercially available bacterial hosts such as SURE® CompetentCells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively,bacterial cells such as E. coli LE392 could be used as host cells forphage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

B. Smad7 Proteins and Protein Fragments

Mothers against decapentaplegic homolog 7 (Smad7) was previouslyidentified as an antagonist of TGF-β signaling by several mechanismsincluding: (a) blockade of TGF-β receptor-mediated phosphorylation andnuclear translocation of signaling Smads; (b) increased degradation ofTGF-β receptors and signaling Smads through specificubiquitin-proteasome pathways and (c) inhibition of signaling Smads fortheir binding to Smad binding elements (SBEs). Smad7 also antagonizesother signaling pathways, like the NF-κB pathway.

Smad7 protein is encoded by the SMAD7 gene, discussed above. Like manyother TGF-β family members, Smad7 is involved in cell signaling. It is aTGF-β type 1 receptor antagonist. It blocks TGF-β1 and activinassociating with the receptor, blocking access to Smad2. It is aninhibitory Smad (I-SMAD) and is enhanced by SMURF2. Smad7 also enhancesmuscle differentiation.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally occurring amino acid polymers as well as aminoacid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, for example, an amino acid analog.As used herein, the terms encompass amino acid chains of any length,including full length proteins, wherein the amino acid residues arelinked by covalent peptide bonds.

In one embodiment, the present technology relates to Smad7 proteincompositions. In addition to the entire Smad7 molecule, the presenttechnology also relates to truncated portions and fragments of thepolypeptide that retain one or more activity associated with Smad7, suchas, but not limited to, increasing proliferation, reducing or inhibitingcell death, reducing excessive inflammation, preventing DNA damage,and/or increasing cell migration, as well as treating or preventing oneor more disease or disorders in which such treatment would be helpful asfurther discussed herein. Such activities can be assessed using one ormore assays including, but not limited to, the ability to blockphosphorylation of Smad2 and/or nuclear translocation of the NF-κB p50subunit, increase cell proliferation, reduce apoptosis and/orradiation-induced DNA damage, reduce inflammation and/or angiogenesis,promote healing in oral mucositis, surgical wounds, diabetes wounds,and/or wounds associated with chronic inflammation in mice.

Protein fragments may be generated by genetic engineering of translationstop sites within the coding region (discussed below). Alternatively,treatment of the Smad7 molecule with proteolytic enzymes, known asproteases, can produces a variety of N-terminal, C-terminal and internalfragments. These fragments may be purified according to known methods,such as precipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

As used herein, reference to an isolated protein or polypeptide in thepresent embodiments include full-length proteins, fusion proteins,chimeric proteins, or any fragment (truncated form, portion) orhomologue of such a protein. More specifically, an isolated protein canbe a protein (including a polypeptide or peptide) that has been removedfrom its natural milieu (i.e., that has been subject to humanmanipulation), and can include, but is not limited to, purifiedproteins, partially purified proteins, recombinantly produced proteins,proteins complexed with lipids, soluble proteins, synthetically producedproteins, and isolated proteins associated with other proteins. As such,“isolated” does not reflect the extent to which the protein has beenpurified. Preferably, an isolated protein is produced recombinantly.

Variants of Smad7 are also provided—these can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein that are not essential for activity,including the truncation mutants described above and herein.Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage and/or translation and/or transcription(protein expression), without the loss of other functions or properties.Substitutions of this kind preferably are conservative, that is, oneamino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, each amino acid can be changed or substituted with a differentamino acid. In making substitutional variants, the hydropathic index,hydrophilicity, charge and size are normally considered.

Specifically contemplated deletion variants of Smad7 include truncationsand fragments, for example, including polypeptide molecules havingN-terminal sequences, but not C-terminal sequences, having C-terminalsequences but not N-terminal sequences, or having internal sequences,but not N-terminal or c-terminal sequences. Specifically contemplatedSmad7 polypeptide truncations or fragments include, but are not limitedto, molecules including amino acid residues 2-258, 259-426, 204-258corresponding to the native human Smad7 protein sequence.

The term “truncated” as used herein in reference to protein sequencesrefers to a molecule that contains the natural N-terminus of acorresponding protein (with or without a cleaved leader sequence), butlacks one or more amino acids starting from the C-terminus of themolecule, or a molecule that contains the natural C-terminus of acorresponding protein (with or without a cleaved leader sequence), butlacks one or more amino acids starting from the N-terminus of themolecule. In some embodiments, molecules lacking at least about 25, atleast about 50, at least about 75, at least about 100, at least about125, at least about 150, at least about 200, at least about 250, atleast about 300, or at least about 350, or at least about 400 aminoacids from one or the other terminus are specifically provided. In someembodiments, a “truncated” molecule is biologically active, having oneor more of the Smad7 activities described herein.

The term “fragment” as used herein in reference to polypeptide sequencesrefers to a molecule containing contiguous residues of a full lengthsequence but lacking some N-terminal and/or C-terminal residues of thefull length sequence. In some embodiments, a “fragment” includes aportion of one or more of the full length sequences described herein. Insome embodiments, the “fragment” does not include sequences encodingeither the N-terminal or the C-terminal, but only internal fragments. Insome embodiments, a “fragment” encodes a polypeptide that isbiologically active, having one or more of the Smad7 activitiesdescribed herein. In some embodiments, polypeptide fragments have atleast about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150 amino acids.

A specialized kind of variant is the fusion protein. This moleculegenerally has all or a substantial portion of the native molecule,linked at the N- or C-terminus, to all or a portion of a secondpolypeptide. However, in some embodiments, the fusion protein mayinclude any one of the fragments and/or truncated (N-terminal,C-terminal) Smad7 proteins described throughout the disclosure. Forexample, fusions may employ leader sequences from other species topermit the recombinant expression of a protein in a heterologous host.Another useful fusion includes the addition of an optional functionallyactive domain, such as but not limited to an antibody epitope and/or apurification tag (e.g., V5: GKPIPNPLLGLDST (SEQ ID NO: 41); Flag:KYKDDDDK (SEQ ID NO: 42); HA: YPYDVPDYA (SEQ ID NO: 43)). Another typeof fusion includes attaching a domain that can act as the target for anactivating or inactivating ligand, thereby permitting control of thefusion protein's function once delivered to a subject. Such domainsinclude, for example, steroid ligand binding (e.g., ER, PR, GR), whichcan be activated by small molecules, e.g., 4-hydroxyl tamoxifen or RU486that are either uniquely able to activate those steroid ligand bindingdomains and/or do not exist in nature and will therefore enable fullcontrol of the Smad7 function by the presence of these small molecules.

Another specific form of a fusion protein finding particular utility inthe present technology is a fusion including a protein transductiondomain (PTD), also called a cell delivery domain or cell transductiondomain. Such domains have been described in the art and are generallycharacterized as short amphipathic or cationic peptides and peptidederivatives, often containing multiple lysine and arginine resides(Fischer, Med. Res. Rev. 27:755-795 (2007)). In some embodiments, thePTD is one or more variant of TAT protein from HIV (GRKKRRQRRR (SEQ IDNO: 2), YGRKKRRQRRR (SEQ ID NO: 4), or GRKKRRQ (SEQ ID NO: 6)) oralternatively, HSV VP 16. Alternate forms of Tat may be used. In someembodiments, a linker may be used to connect one or more PTDs and SMad7.In some embodiments, the PTD (optionally Tat) is fused or linked inframe to the N-terminal and/or C-terminal end of any one of the Smad7full-length, fragments, and/or truncated (N-terminal, C-terminal)proteins described throughout the disclosure. Other examples of PTDsprovided by the present technology are shown in Table 1.

TABLE 1 PROTEIN TRANSDUCTION DOMAINS SEQ ID NO: GALFLGWLGAAGSTMGAKKKRKV44 RQIKIWFQNRRMKWKK 45 RRMKWKK 46 RRWRRWWRRWWRRWRR 47 RGGRLSYSRRRFSTSTGR48 YGRKKRRQRRR  4 RKKRRQRRR 49 YARAAARQARA 50 RRRRRRRR 51 KKKKKKKK 52GWTLNSAGYLLGKINLKALAALAKXIL 53 LLILLRRRIRKQANAHSK 54 SRRHHCRSKAKRSRHH 55NRARRNRRRVR 56 RQLRIAGRRLRGRSR 57 KLIKGRTPIKFGK 58 RRIPNRRPRR 59KLALKLALKALKAALKLA 60 KLAKLAKKLAKLAK 61 GALFLGFLGAAGSTNGAWSQPKKKRKV 62KETWWETWWTEWSQPKKKRKV 63 LKKLLKKLLKKLLKKLLKKL 64QAATATRGRSAASRPTERPRAPARSASRPRRPVE 65 MGLGLHLLVLAAALQGAKSKRKV 66AAVALLPAVLLALLAPAAANYKKPKL 67 MANLGYWLLALFVTMWTDVGLCKKRPKP 68LGTYTQDFNKFHTFPQTAIGVGAP 69 DPKGDPKGVTVTVTVTVTGKGDPXPD 70 PPPPPPPPPPPPPP71 VRLPPPVRLPPPVRLPPP 72 PRPLPPPRPG 73 SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 74TRSSRAGLQFPVGRVHRLLRK 75 GIGKFLHSAKKFGKAFVGEIMNS 76KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 77 ALWMTLLKKVLKAAAKAALNAVLVGANA 78GIGAVLKVLTTGLPALISWIKRKRQQ 79 INLKALAALAKKIL 80GFFALIPKIISSPLPKTLLSAVGSALGGSGGQE 81 LAKWALKQGFAKLKS 82SMAQDIISTIGDLVKWIIQTVNXFTKK 83 LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPRTES84 PAWRKAFRWAWRMLKKAA 85 KLKLKLKLKLKLKLKLKL 86

In particular embodiments, the present technology provides for sequencevariants of Smad7 in which one or more residues have been altered. Forexample, in one embodiment, the methionine residue found at position 216of the human Smad7 sequence is modified to a leucine residue (ATG toCTG).

C. Methods of Treatment

Smad7-treatable diseases and disorders may include those including oneor more of reduced cell proliferation, reduced cell migration, increasedcell death, excessive inflammation, and/or DNA damage. Smad7-relateddiseases and disorders may include those where treatment with a Smad7protein and biologically active fragments and derivatives thereof thathave one or more activities including but not limited to increasingproliferation, reducing or inhibiting cell death, reducing excessiveinflammation, preventing DNA damage, and/or increasing cell migration ishelpful. Such diseases and/or disorders may include but are not limitedto acute (e.g., through surgery, combat, trauma) and chronic wounds(e.g., ulcers, such as diabetic, pressure, venous), scarring, fibrosis,and aberrant healing, mucositis (e.g., oral and/or gastro-intestinal),stomatitis, proctitis, autoimmune disease (e.g., psoriasis, arthritis),and cancer.

In some embodiments, one or more of the diseases and or disordersdescribed herein may be prevented, treated, and/or ameliorated byproviding to a subject in need of such treatment a therapeuticallyeffective amount of one or more of the Smad7 proteins (e.g., full-lengthor biologically active truncated (e.g., N-terminal or C-terminal) orfragment thereof) described in the disclosure. In some embodiments, theone or more Smad7 proteins are fusion proteins including a PTD domain.In some embodiments, the one or more Smad7 proteins includes Leu216. Insome embodiments, the Smad7 proteins make part of a pharmaceuticalcomposition including one or more pharmaceutically acceptableexcipients.

In some embodiments, one or more of the diseases and or disordersdescribed herein may be prevented, treated, and/or ameliorated byproviding to a subject in need of such treatment a therapeuticallyeffective amount of one or more of the nucleic acid molecules encodingone or more Smad7 proteins (e.g., full-length or biologically activetruncated (e.g., N-terminal or C-terminal) or fragment thereof)described in the disclosure. In some embodiments, the one or morenucleic acid molecules include codon-optimized nucleotide sequencesand/or sequences that encode Leu216. In some embodiments, the one ormore Smad7 nucleic acid molecules are provided to the subject in aconstruct including an expression vector. In some embodiments, the Smad7nucleic acid molecules (optionally part of an expression vector) makepart of a pharmaceutical composition including one or morepharmaceutically acceptable excipients.

The term “subject” or “patient” as used herein refers to persons ornon-human animals in need of treatment and or prevention using one ormore of the treatments described herein. In some embodiments, non-humananimals include laboratory animals such as monkeys, mice, rats, andrabbits, domestic pets such as dogs and cats, and livestock such ascattle, horses, pigs, goats and sheep.

1. Chronic Wounds

A chronic wound is a wound that does not heal in an orderly set ofstages and in a predictable amount of time the way most wounds do;wounds that do not heal within three months are often consideredchronic. Chronic wounds seem to be detained in one or more of the phasesof wound healing. For example, chronic wounds often remain in theinflammatory stage for too long. In acute wounds, there is a precisebalance between production and degradation of molecules such ascollagen; in chronic wounds this balance is lost and degradation playstoo large a role.

As described in more detail elsewhere herein, PTD-Smad7 has been shownto enhance wound healing in a mouse skin model and a mucosal model.Application of PTD-Smad7 was effective through a topical route, which isdesirable for wound treatment. Although not intending to be bound bytheory, it is believed that PTD-Smad7 may act to treat or amelioratechronic wounds through multiple routes, which may include one or more ofreducing inflammation, increasing cell proliferation (e.g.,keratinocytes), increasing cell migration (e.g., keratinocytes), orreducing fibrosis (e.g., through modulation of collagen), among others.

Chronic wounds may never heal or may take years to do so. These woundscause patients severe emotional and physical stress as well as creatinga significant financial burden on patients and the whole healthcaresystem. Acute and chronic wounds are at opposite ends of a spectrum ofwound healing types that progress toward being healed at differentrates. The vast majority of chronic wounds can be classified into threecategories: venous ulcers, diabetic, and pressure ulcers. A small numberof wounds that do not fall into these categories may be due to causessuch as radiation poisoning or ischemia.

Venous and Arterial Ulcers.

Venous ulcers, which usually occur in the legs, account for about 70% to90% of chronic wounds and mostly affect the elderly. They are thought tobe due to venous hypertension caused by improper function of valves thatexist in the veins to prevent blood from flowing backward. Ischemiaresults from the dysfunction and, combined with reperfusion injury,causes the tissue damage that leads to the wounds.

Diabetic Ulcers.

Another major cause of chronic wounds, diabetes, is increasing inprevalence. Diabetics have a 15% higher risk for amputation than thegeneral population due to chronic ulcers. Diabetes causes neuropathy,which inhibits nociception and the perception of pain. Thus patients maynot initially notice small wounds to legs and feet, and may thereforefail to prevent infection or repeated injury. Further, diabetes causesimmune compromise and damage to small blood vessels, preventing adequateoxygenation of tissue, which can cause chronic wounds. Pressure alsoplays a role in the formation of diabetic ulcers.

Pressure Ulcers.

Another leading type of chronic wounds is pressure ulcers, which usuallyoccur in people with conditions such as paralysis that inhibit movementof body parts that are commonly subjected to pressure such as the heels,shoulder blades, and sacrum. Pressure ulcers are caused by ischemia thatoccurs when pressure on the tissue is greater than the pressure incapillaries, and thus restricts blood flow into the area. Muscle tissue,which needs more oxygen and nutrients than skin does, shows the worsteffects from prolonged pressure. As in other chronic ulcers, reperfusioninjury damages tissue.

Chronic wounds may affect only the epidermis and dermis, or they mayaffect tissues all the way to the fascia. They may be formed originallyby the same things that cause acute wounds, such as surgery oraccidental trauma, or they may form as the result of systemic infection,vascular, immune, or nerve insufficiency, or comorbidities such asneoplasias or metabolic disorders. Although not intending to be bound bytheory, the reason a wound becomes chronic is that the body's ability todeal with the damage is overwhelmed by factors such as repeated trauma,continued pressure, ischemia, or illness. Some of the major factors thatlead to chronic wounds include, but are not limited to, ischemia,reperfusion injury, and bacterial colonization.

Ischemia.

Ischemia is an important factor in the formation and persistence ofwounds, especially when it occurs repetitively (as it usually does) orwhen combined with a patient's old age. Ischemia causes tissue to becomeinflamed and cells to release factors that attract neutrophils such asinterleukins, chemokines, leukotrienes, and complement factors.

While they fight pathogens, neutrophils also release inflammatorycytokines and enzymes that damage cells. One of their importantfunctions is to produce Reactive Oxygen Species (ROS) to kill bacteria,for which they use an enzyme called myeloperoxidase. The enzymes and ROSproduced by neutrophils and other leukocytes damage cells and preventcell proliferation and wound closure by damaging DNA, lipids, proteins,the ECM, and cytokines that speed healing. Neutrophils remain in chronicwounds for longer than they do in acute wounds, and contribute to thefact that chronic wounds have higher levels of inflammatory cytokinesand ROS. Because wound fluid from chronic wounds has an excess ofproteases and ROS, the fluid itself can inhibit healing by inhibitingcell growth and breaking down growth factors and proteins in the ECM.

Bacterial colonization. Since more oxygen in the wound environmentallows white blood cells to produce ROS to kill bacteria, patients withinadequate tissue oxygenation, for example, those who sufferedhypothermia during surgery, are at higher risk for infection. The host'simmune response to the presence of bacteria prolongs inflammation,delays healing, and damages tissue. Infection can lead not only tochronic wounds but also to gangrene, loss of the infected limb, anddeath of the patient.

Like ischemia, bacterial colonization and infection damage tissue bycausing a greater number of neutrophils to enter the wound site. Inpatients with chronic wounds, bacteria with resistance to antibioticsmay have time to develop. In addition, patients carrying drug resistantbacterial strains, such as methicillin-resistant Staphylococcus aureus(MRSA), have more chronic wounds.

Growth Factors and Proteolytic Enzymes.

Chronic wounds also differ in makeup from acute wounds in that theirlevels of proteolytic enzymes such as elastase and matrixmetalloproteinases (MMPs) are higher, while their concentrations ofgrowth factors such as Platelet-derived growth factor and KeratinocyteGrowth Factor are lower.

Since growth factors (GFs) are imperative in timely wound healing,inadequate GF levels may be an important factor in chronic woundformation. In chronic wounds, the formation and release of growthfactors may be prevented, the factors may be sequestered and unable toperform their metabolic roles, or degraded in excess by cellular orbacterial proteases.

Chronic wounds such as diabetic and venous ulcers are also caused by afailure of fibroblasts to produce adequate ECM proteins and bykeratinocytes to epithelialize the wound. Fibroblast gene expression isdifferent in chronic wounds than in acute wounds.

Although all wounds require a certain level of elastase and proteasesfor proper healing, too high a concentration is damaging. Leukocytes inthe wound area release elastase, which increases inflammation, destroystissue, proteoglycans, and collagen, and damages growth factors,fibronectin, and factors that inhibit proteases. The activity ofelastase is increased by human serum albumin, which is the most abundantprotein found in chronic wounds. However, chronic wounds with inadequatealbumin are especially unlikely to heal, so regulating the wound'slevels of that protein may in the future prove helpful in healingchronic wounds.

Excess matrix metalloproteinases, which are released by leukocytes, mayalso cause wounds to become chronic. MMPs break down ECM molecules,growth factors, and protease inhibitors, and thus increase degradationwhile reducing construction, throwing the delicate compromise betweenproduction and degradation out of balance.

Oral Ulcers.

A mouth ulcer (also termed an oral ulcer, or a mucosal ulcer) is anulcer that occurs on the mucous membrane of the oral cavity. Moreplainly, a mouth ulcer is a sore or open lesion in the mouth. Mouthulcers are very common, occurring in association with many diseases andby many different mechanisms, but usually there is no serious underlyingcause. The two most common causes of oral ulceration are local trauma(e.g., rubbing from a sharp edge on a filling) and aphthous stomatitis(“canker sores”), a condition characterized by recurrent formation oforal ulcers for largely unknown reasons. Some consider ulcers on thelips or on the skin around the mouth to be included under the generalterm oral ulceration (e.g., an ulcer left by rupture of a blister causedby herpes labialis, i.e., a cold sore). Mouth ulcers often cause painand discomfort, and may alter the person's choice of food while healingoccurs (e.g., avoiding acidic or spicy foods and beverages). They mayoccur singly or multiple ulcers may occur at the same time (a “crop” ofulcers). Once formed, the ulcer may be maintained by inflammation and/orsecondary infection. Rarely, a mouth ulcer that does not heal for manyweeks may be a sign of oral cancer. Other causes include burns, chemicalinjury, or infection.

A mucosal ulcer is an ulcer which specifically occurs on a mucousmembrane. An ulcer is a tissue defect which has penetrated theepithelial-connective tissue border, with its base at a deep level inthe submucosa, or even within muscle or periosteum. An ulcer is a deeperbreech of the epithelium than an erosion or an excoriation, and involvesdamage to both epithelium and lamina propria. An erosion is asuperficial breach of the epithelium, with little damage to theunderlying lamina propria. A mucosal erosion is an erosion whichspecifically occurs on a mucous membrane. Only the superficialepithelial cells of the epidermis or of the mucosa are lost, and thelesion can reach the depth of the basement membrane. Erosions healwithout scar formation. Excoriation is a term sometimes used to describea breach of the epithelium which is deeper than an erosion but shallowerthan an ulcer. This type of lesion is tangential to the rete pegs andshows punctiform (small pinhead spots) bleeding, caused by exposedcapillary loops.

2. Acute Wounds/Trauma

Physical trauma is a serious and body-altering physical injury, such asthe removal of a limb. Blunt force trauma, a type of physical traumacaused by impact or other force applied from or with a blunt object,whereas penetrating trauma is a type of physical trauma in which theskin or tissues are pierced by an object. Trauma can also be describedas both unplanned, such as an accident, or planned, in the case ofsurgery. Both can be characterized by mild to severe tissue damage,blood loss and/or shock, and both may lead to subsequent infection,including sepsis. The present technology provides for treatment oftrauma, including both pre-treatment (in the case of a medicalprocedure) and treatment after trauma injury has occurred.

As described in more detail elsewhere herein (and briefly mentionedabove), PTD-Smad7 has been shown to enhance wound healing in a mouseskin model and a mucosal model. Application of PTD-Smad7 was effectivethrough a topical route, which is desirable for wound treatment.Although not intending to be bound by theory, it is believed thatPTD-Smad7 may act to treat or ameliorate wounds through multiple routes,which may include one or more of reducing inflammation, increasing cellproliferation (e.g., keratinocytes), increasing cell migration (e.g.,keratinocytes), or reducing fibrosis (e.g., through modulation ofcollagen), among others. As described briefly below, reducedinflammation could significantly contribute to accelerated woundhealing, optionally through reduced angiogenesis and collagen productionand/or reduced leukocyte infiltration leading to reduction of cytokinesand chemokines normally released by leukocytes, which are angiogenic andfibrogenic. Temporal treatment with Smad7 may allow early stageangiogenesis and collagen production required for wound repair, whilepreventing prolonged angiogenesis and collagen production. These changescould potentially accelerate wound stromal remodeling and preventexcessive scarring due to unresolved inflammation or collagenoverproduction. For surgical procedures (as well as everyday injuries),particularly where the potential for scarring is an issue, treatmentwith Smad7 may be beneficial.

Surgery.

Surgery uses operative manual and instrumental techniques on a patientto investigate and/or treat a pathological condition such as disease orinjury, to help improve bodily function or appearance, or sometimes forsome other reason. The present technology can address trauma resultingfrom surgeries, as defined further below.

As a general rule, a procedure is considered surgical when it involvescutting of a patient's tissues or closure of a previously sustainedwound. Other procedures that do not necessarily fall under this rubric,such as angioplasty or endoscopy, may be considered surgery if theyinvolve common surgical procedure or settings, such as use of a sterileenvironment, anesthesia, antiseptic conditions, typical surgicalinstruments, and suturing or stapling. All forms of surgery areconsidered invasive procedures; so-called noninvasive surgery usuallyrefers to an excision that does not penetrate the structure beingaddressed (e.g., laser ablation of the cornea) or to a radiosurgicalprocedure (e.g., irradiation of a tumor). Surgery can last from minutesto hours.

Surgical procedures are commonly categorized by urgency, type ofprocedure, body system involved, degree of invasiveness, and specialinstrumentation. Elective surgery is done to correct anon-life-threatening condition, and is carried out at the patient'srequest, subject to the surgeon's and the surgical facility'savailability. Emergency surgery is surgery which must be done quickly tosave life, limb, or functional capacity. Exploratory surgery isperformed to aid or confirm a diagnosis. Therapeutic surgery treats apreviously diagnosed condition.

Amputation involves cutting off a body part, usually a limb or digit.Replantation involves reattaching a severed body part. Reconstructivesurgery involves reconstruction of an injured, mutilated, or deformedpart of the body. Cosmetic surgery is done to improve the appearance ofan otherwise normal structure. Excision is the cutting out of an organ,tissue, or other body part from the patient. Transplant surgery is thereplacement of an organ or body part by insertion of another fromdifferent human (or animal) into the patient. Removing an organ or bodypart from a live human or animal for use in transplant is also a type ofsurgery.

When surgery is performed on one organ system or structure, it may beclassified by the organ, organ system or tissue involved. Examplesinclude cardiac surgery (performed on the heart), gastrointestinalsurgery (performed within the digestive tract and its accessory organs),and orthopedic surgery (performed on bones and/or muscles).

Minimally invasive surgery involves smaller outer incision(s) to insertminiaturized instruments within a body cavity or structure, as inlaparoscopic surgery or angioplasty. By contrast, an open surgicalprocedure requires a large incision to access the area of interest.Laser surgery involves use of a laser for cutting tissue instead of ascalpel or similar surgical instruments. Microsurgery involves the useof an operating microscope for the surgeon to see small structures.Robotic surgery makes use of a surgical robot, such as Da Vinci or Zeussurgical systems, to control the instrumentation under the direction ofthe surgeon.

3. Autoimmune/Inflammatory Disease

The present technology contemplates the treatment of a variety ofautoimmune and/or inflammatory disease states such asspondyloarthropathy, ankylosing spondylitis, psoriatic arthritis,reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn'sdisease, irritable bowel disease, inflammatory bowel disease, rheumatoidarthritis, juvenile rheumatoid arthritis, familial Mediterranean fever,amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis,viral arthritis, multiple sclerosis, or psoriasis. The diagnosis andtreatment of these diseases are well documented in the literature.

In general, autoimmune diseases are associated with an overactive immuneresponse of a body against substances and tissues normally present inthe body, and not normally the focus of an immune response. There aremore than 80 types of autoimmune diseases, some of which have similarsymptoms, and they may arise from a similar underlying cause. Theclassic sign of an autoimmune disease is inflammation, which asdisclosed herein is amenable to treatment with Smad7 (optionallyPTD-Smad7) compositions.

4. Chemotherapy, Radiotherapy and Cytokine Therapy Toxicity

Various forms of cancer therapy, including chemotherapy, radiation, andcytokines, are associated with toxicity, sometimes severe, in the cancerpatient. The present technology seeks to reduce this toxicity using thepharmaceutical compositions of the present technology, thereby reducingor alleviating discomfort on the part of the patient, as well aspermitting higher doses of the therapy.

As described at length throughout this disclosure, it has been foundthat PTD-Smad7 acts to heal as well as to prevent oral mucositis in amouse model. PTD-Smad7 was shown to be more effective than palifermin,the existing approved drug for preventing oral mucositis, in directcomparisons.

Oral cancer, the 6^(th) most common cancer worldwide, is a subtype ofhead and neck cancer, and includes any cancerous tissue growth locatedin the oral cavity. It may arise as a primary lesion originating in anyof the oral tissues, by metastasis from a distant site of origin, or byextension from a neighboring anatomic structure, such as the nasalcavity or the oral cancers may originate in any of the tissues of themouth, and may be of varied histologic types: teratoma, adenocarcinomaderived from a major or minor salivary gland, lymphoma from tonsillar orother lymphoid tissue, or melanoma from the pigment-producing cells ofthe oral mucosa. There are several types of oral cancers, but around 90%are squamous cell carcinomas, originating in the tissues that line themouth and lips. Oral or mouth cancer most commonly involves the tongue.It may also occur on the floor of the mouth, cheek lining, gingiva(gums), lips, or palate (roof of the mouth). Most oral cancers look verysimilar under the microscope and are called squamous cell carcinoma.These are malignant and tend to spread rapidly.

Over 80% of oral cancer patients are treated with radiation therapy andat least 75% of these individuals will develop oral mucositis. Oralmucositis is a chronic oral ulceration. This disease frequently occursin radiation-treated patients of all cancer types, including but notlimited to patients who are radiation-treated for organ transplants (toeliminate rejection of the transplants), and patients undergoing routinechemotherapy. Severe oral mucositis is extremely painful and impairsfood/liquid intake, hence is often the most severe complication ofcancer therapy. Oral mucositis is a major factor in determining themaximum dose possible of radiation and chemotherapy to the head and neckregion; it can significantly complicate cancer treatment, extendhospitalization, decrease quality of life and increase costs.

Currently, there is no established therapy to effectively treat severeoral mucositis. To date, palifermin (KEPIVANCE®), a recombinant proteinof human keratinocyte growth factor (KGF), is the only FDA approved drugfor intravenous (i.v.) injections for severe oral mucositis inbone-marrow transplant patients, and its use in cancer patients remainsto be determined. It is also used for prevention of oral mucositis.Hence, this drug is available for only 4% of the at-risk population. Italso suffers from the need for medical service providers due to the i.v.administration route. Other potential therapies include topical rinses,such as viscous 2% lidocaine rinses, or baking soda and salinesolutions, or a cocktail solution, for instance BAX (lidocaine,diphenhyramine, sorbitol and MYLANTA®). Other investigative ormucoprotective adjuvant therapies include, but are not limited to, betacarotene, tocopherol, laser irradiation, prophylactic brushing the oralmucosa with silver-nitrate, misoprostol, leucovorin, systemic KGF,pentoxifylline, allopurinol mouthwash, systemic sucralfate,chlorhexidine gluconate, and cryotherapy.

Chemotherapy- and radiation-induced gut mucositis is an inflammatorycondition that arises as a result of the acute death of rapidly dividingintestinal epithelial cells. Most chemotherapeutic drugs used fortreatment of solid tumors, alone, in a combination of drugs, or withradiation, will result in the death of a large number of intestinalepithelial cells. The clinical manifestations of the ensuing mucositisinclude digestive symptoms such as nausea and vomiting, seriousdiarrhea, acute weight loss and wasting. This is fast becoming one ofthe limiting factors for administering chemotherapy for many cancerpatients. The ability of Tat-Smad7 to protect intestinal epithelialcells from either chemotherapeutic agents, radiation, or a combinationsof those, will significantly decrease the undesirable side effects ofcancer therapies, and enable more aggressive ways to treat the diseasewith existing tools.

Bone marrow failure syndromes are a set of conditions that develop whenthe hematopoietic stem cell compartment is compromised and fails to giverise to normal cell types. Bone marrow failure occurs as a result ofinherited genetic abnormalities, exposure to a noxious substance, suchas toxins, chemicals or viruses. Although the nature and identity ofenvironmental factors that can lead to the development of acquired bonemarrow failure is still not completely understood, a few factors havebeen linked to the development of acquired bone marrow failure amongmilitary personnel including exposure to mustard gas, ionizingradiation, and infectious agents such as visceral leishmaniasis orAfrican trypanosomiasis. The best approach for management of bone marrowfailure syndromes is still the transplantation of hematopoietic stemcells (HSCs), unless a sufficient number of the remaining resident bonemarrow HSCs can be spared from these stresses and encouraged torepopulate the hematopoietic compartment. The modulation of Smad 7, asdescribed here, should enable for the deliberate protection of theremaining resident HSCs in patients that exhibit clinical signsconsistent with bone marrow failure.

5. Cancer

TGF-β and NF-κB activations are known to promote cancer invasion andmetastasis. Currently, TGF-β inhibitors are in clinical trials fortreating metastatic cancer and NF-κB inhibitors are used in cancerprevention. The demonstrated effect of Smad7 on blocking both TGF-3 andNF-κB signaling present the possibility that it is an even strongeranti-cancer/anti-metastasis agent than other inhibitors that inhibitonly one of these two pathways. Smad7 has been shown to preventangiogenesis and fibrogenesis, and may therefore be particularly usefulin situations where the tumor needs to develop a blood supply and/orstroma.

The cancer may be selected from the group consisting of brain, lung,liver, spleen, kidney, lymph node, small intestine, pancreas, bloodcells, colon, stomach, breast, endometrium, prostate, testicle, cervix,uterus, ovary, skin, head & neck, esophagus, bone marrow and bloodcancer. The cancers may be metastatic or primary, recurrent ormulti-drug resistant. In some embodiments, the cancer is a solid tumor(organ tumor). Solid tumors refer to a mass of cells that grow in organsystems and can occur anywhere in the body. Two types of solid tumorsinclude epithelial tumors (carcinomas) that occur in the epithelialtissue inside or outside an organ, and sarcomas (connective tissuetumors) that occur in connective tissue such as, but not limited to,muscles, tendons, fat, nerves and other connective tissues that support,surround, or connect structures and organs in the body. In someembodiments the cancer is a liquid tumor or cancer of the blood, bonemarrow, or lymph nodes. These tumors include, but are not limited to,leukemia, lymphoma, and myeloma.

6. Scarring, Fibrosis, and Aberrant Healing

In addition to accelerated re-epithelialization (e.g., throughincreasing cell proliferation and/or increasing cell migration), Smad7effects on wound stroma include one or more of reducing inflammation,angiogenesis, or collagen production, among others. Although notintending to be bound by theory these effects may be mediated throughreduction of NF-κB signaling (evidenced by reduced p50), and blockingTGF-β signaling (evidenced by reduced pSmad2). As a result, reducedinflammation could significantly contribute to accelerated woundhealing, optionally through reduced angiogenesis and collagen productionand/or reduced leukocyte infiltration leading to reduction of cytokinesand chemokines normally released by leukocytes, which are angiogenic andfibrogenic. Temporal treatment with Smad7 may allow early stageangiogenesis and collagen production required for wound repair, whilepreventing prolonged angiogenesis and collagen production. These changescould potentially accelerate wound stromal remodeling and preventexcessive scarring due to unresolved inflammation or collagenoverproduction.

7. Stomatitis

Stomatitis is an inflammation of the mucous lining of any of thestructures in the mouth, which may involve the cheeks, gums, tongue,lips, throat, and roof or floor of the mouth. The inflammation can becaused by conditions in the mouth itself, such as poor oral hygiene,dietary protein deficiency, poorly fitted dentures, or from mouth burnsfrom hot food or drinks, toxic plants, or by conditions that affect theentire body, such as medications, allergic reactions, radiation therapy,or infections. Severe iron deficiency anemia can lead to stomatitis.Iron is necessary for the upregulation of transcriptional elements forcell replication and repair. Lack of iron can cause the geneticdownregulation of these elements, leading to ineffective repair andregeneration of epithelial cells, especially in the mouth and lips. Thiscondition is also prevalent in people who have a deficiency in vitaminB₂ (Riboflavin), B₃ (Niacin), B₆ (Pyridoxine), B₉ (folic acid) or B₁₂(cobalamine) When it also involves an inflammation of the gingiva(gums), it is called gingivostomatitis. It may also be seen inariboflavinosis (riboflavin deficiency) or neutropenia.

Irritation and fissuring in the corners of the lips is termed angularstomatitis or angular cheilitis. In children, angular stomatitis is afrequent cause is repeated lip-licking and in adults it may be a sign ofunderlying iron deficiency anemia, or vitamin B deficiencies (e.g.,B₂-riboflavin, B₉-folate or B₁₂-cobalamin), which in turn may beevidence of poor diets or malnutrition (e.g., celiac disease). Also,angular cheilitis can be caused by a patient's jaws at rest being“overclosed” due to edentulousness or tooth wear, causing the jaws tocome to rest closer together than if the complete/unaffected dentitionwere present. This causes skin folds around the angle of the mouth whichare kept moist by saliva which in turn favours infection; mostly byCandida albicans or similar species. Treatment usually involves theadministration of topical nystatin or similar antifungal agents. Anothertreatment can be to correct the jaw relationship with dental treatment(e.g., dentures or occlusal adjustment).

Migratory stomatitis is a condition in which extensive areas in the oralcavity mucosa are affected by annular atrophic red lesions that aresurrounded by a thin white rim. This is a relatively uncommon form ofthe geographic tongue condition, that, as opposed to migratorystomatitis, is confined to the dorsal and lateral aspects of the tonguemucosa only.

8. Proctitis

Proctitis is inflammation of the lining of the rectum, the lower end ofthe large intestine leading to the anus. With proctitis, inflammation ofthe rectal lining—called the rectal mucosa—is uncomfortable andsometimes painful. The condition may lead to bleeding or mucousdischarge from the rectum, among other symptoms. Some causes ofproctitis include, but are not limited to: sexually transmitted diseases(STDs), such as those that can be transmitted during anal sex (e.g.,gonorrhea, chlamydia, syphilis, and herpes); non-STD infections from,for example, food borne bacteria (e.g., Salmonella and Shigella);anorectal trauma from, for example, anal sex or the insertion of objectsor substances into the rectum (e.g., chemicals from enemas); ulcerativecolitis and Crohn's disease or other inflammatory bowel diseases, maycause ulcers (e.g., sores) in the inner lining of the colon and rectum;radiation therapy, particularly of the pelvic area (e.g., rectal,ovarian, or prostate cancer) which may lead to rectal bleeding;antibiotics which lead to a loss of commensal bacteria allowing harmfulbacteria (e.g., Clostridium difficile) to cause disease.

9. Formulations and Routes of Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—proteins, expression vectors, virusstocks, proteins and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

PTD-Smad7 (and truncated variants) were purified extensively prior touse in animal models. PTD-Smad7 (and truncated versions) were preparedfor topical and trans-mucosal application using a mixture of glyceroland PBS.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present technology comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present technology, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present technology may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present technology will be via any common route so longas the target tissue is available via that route. Such routes ofadministration may include oral parenteral (including intravenous,intramuscular, subcutaneous, intradermal, intra-articular,intra-synovial, intrathecal, intra-arterial, intracardiac, subcutaneous,intraorbital, intracapsular, intraspinal, intrastemal, and transdermal),nasal, buccal, urethral, rectal, vaginal, mucosal, dermal, or topical(including dermal, buccal, and sublingual). Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. Of particular interest isdirect intratumoral administration, perfusion of a tumor, oradministration local or regional to a tumor, for example, in the localor regional vasculature or lymphatic system, or in a resected tumor bed.Administration can also be via nasal spray, surgical implant, internalsurgical paint, infusion pump, or via catheter, stent, balloon or otherdelivery device. The most useful and/or beneficial mode ofadministration can vary, especially depending upon the condition of therecipient and the disorder being treated.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The compositions of the present technology may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The formulations are easily administered in a variety of dosage forms.Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

For oral administration the polypeptides of the present technology maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. It is anticipated that virtually any pillor capsule type known to one of skill in the art including, e.g.,coated, and time delay, slow release, etc., may be used with the presenttechnology. A mouthwash may be prepared incorporating the activeingredient in the required amount in an appropriate solvent, such as asodium borate solution (Dobell's Solution). Alternatively, the activeingredient may be incorporated into an antiseptic wash containing sodiumborate, glycerin and potassium bicarbonate. The active ingredient mayalso be dispersed in dentifrices, including: gels, pastes, creams,powders and slurries. The active ingredient may be added in atherapeutically effective amount to a paste dentifrice that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants.

Pharmaceutical compositions suitable for oral dosage may take variousforms, such as tablets, capsules, caplets, and wafers (including rapidlydissolving or effervescing), each containing a predetermined amount ofthe active agent. The compositions may also be in the form of a powderor granules, a solution or suspension in an aqueous or non-aqueousliquid, and as a liquid emulsion (oil-in-water and water-in-oil). Theactive agents may also be delivered as a bolus, electuary, or paste. Itis generally understood that methods of preparations of the above dosageforms are generally known in the art, and any such method would besuitable for the preparation of the respective dosage forms for use indelivery of the compositions.

In one embodiment, an active agent compound may be administered orallyin combination with a pharmaceutically acceptable vehicle such as aninert diluent or an edible carrier. Oral compositions may be enclosed inhard or soft shell gelatin capsules, may be compressed into tablets ormay be incorporated directly with the food of the patient's diet. Thepercentage of the composition and preparations may be varied; however,the amount of substance in such therapeutically useful compositions ispreferably such that an effective dosage level will be obtained.

Hard capsules containing the active agent compounds may be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the compound, and may further comprise additionalingredients including, for example, an inert solid diluent such ascalcium carbonate, calcium phosphate, or kaolin. Soft gelatin capsulescontaining the compound may be made using a physiologically degradablecomposition, such as gelatin. Such soft capsules comprise the compound,which may be mixed with water or an oil medium such as peanut oil,liquid paraffin, or olive oil.

Sublingual tablets are designed to dissolve very rapidly. Examples ofsuch compositions include ergotamine tartrate, isosorbide dinitrate, andisoproterenol HCL. The compositions of these tablets contain, inaddition to the drug, various soluble excipients, such as lactose,powdered sucrose, dextrose, and mannitol. The solid dosage forms of thepresent technology may optionally be coated, and examples of suitablecoating materials include, but are not limited to, cellulose polymers(such as cellulose acetate phthalate, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,and hydroxypropyl methylcellulose acetate succinate), polyvinyl acetatephthalate, acrylic acid polymers and copolymers, and methacrylic resins(such as those commercially available under the trade name EUDRAGIT®),zein, shellac, and polysaccharides.

Powdered and granular compositions of a pharmaceutical preparation maybe prepared using known methods. Such compositions may be administereddirectly to a patient or used in the preparation of further dosageforms, such as to form tablets, fill capsules, or prepare an aqueous oroily suspension or solution by addition of an aqueous or oily vehiclethereto. Each of these compositions may further comprise one or moreadditives, such as dispersing or wetting agents, suspending agents, andpreservatives. Additional excipients (e.g., fillers, sweeteners,flavoring, or coloring agents) may also be included in thesecompositions.

Liquid compositions of pharmaceutical compositions which are suitablefor oral administration may be prepared, packaged, and sold either inliquid form or in the form of a dry product intended for reconstitutionwith water or another suitable vehicle prior to use.

A tablet containing one or more active agent compounds described hereinmay be manufactured by any standard process readily known to one ofskill in the art, such as, for example, by compression or molding,optionally with one or more adjuvant or accessory ingredient. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active agents.

Solid dosage forms may be formulated so as to provide a delayed releaseof the active agents, such as by application of a coating. Delayedrelease coatings are known in the art, and dosage forms containing suchmay be prepared by any known suitable method. Such methods generallyinclude that, after preparation of the solid dosage form (e.g., a tabletor caplet), a delayed release coating composition is applied.Application can be by methods, such as airless spraying, fluidized bedcoating, use of a coating pan, or the like. Materials for use as adelayed release coating can be polymeric in nature, such as cellulosicmaterial (e.g., cellulose butyrate phthalate, hydroxypropylmethylcellulose phthalate, and carboxymethyl ethylcellulose), andpolymers and copolymers of acrylic acid, methacrylic acid, and estersthereof.

Solid dosage forms according to the present technology may also besustained release (i.e., releasing the active agents over a prolongedperiod of time), and may or may not also be delayed release. Sustainedrelease compositions are known in the art and are generally prepared bydispersing a drug within a matrix of a gradually degradable orhydrolyzable material, such as an insoluble plastic, a hydrophilicpolymer, or a fatty compound. Alternatively, a solid dosage form may becoated with such a material.

Compositions for parenteral administration include aqueous andnon-aqueous sterile injection solutions, which may further containadditional agents, such as antioxidants, buffers, bacteriostats, andsolutes, which render the compositions isotonic with the blood of theintended recipient. The compositions may include aqueous and non-aqueoussterile suspensions, which contain suspending agents and thickeningagents. Such compositions for parenteral administration may be presentedin unit-dose or multi-dose containers, such as, for example, sealedampoules and vials, and may be stores in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, water (for injection), immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets of the kind previously described.

Compositions for rectal delivery include rectal suppositories, creams,ointments, and liquids. Suppositories may be presented as the activeagents in combination with a carrier generally known in the art, such aspolyethylene glycol. Such dosage forms may be designed to disintegraterapidly or over an extended period of time, and the time to completedisintegration can range from a short time, such as about 10 minutes, toan extended period of time, such as about 6 hours.

Topical compositions may be in any form suitable and readily known inthe art for delivery of active agents to the body surface, includingdermally, buccally, and sublingually. Typical examples of topicalcompositions include ointments, creams, gels, pastes, and solutions.Compositions for administration in the mouth include lozenges.

In accordance with these embodiments, oral (topical, mucosal, and/ordermal) delivery materials can also include creams, salves, ointments,patches, liposomes, nanoparticles, microparticles, timed-releaseformulations and other materials known in the art for delivery to theoral cavity, mucosa, and/or to the skin of a subject for treatmentand/or prevention of a condition disclosed herein. Certain embodimentsconcern the use of a biodegradable oral (topical, mucosal, and/ordermal) patch delivery system or gelatinous material. These compositionscan be a liquid formulation or a pharmaceutically acceptable deliverysystem treated with a formulation of these compositions, and may alsoinclude activator/inducers.

The compositions for use in the methods of the present technology mayalso be administered transdermally, wherein the active agents areincorporated into a laminated structure (generally referred to as a“patch”) that is adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Typically,such patches are available as single layer “drug-in-adhesive” patches oras multi-layer patches where the active agents are contained in a layerseparate from the adhesive layer. Both types of patches also generallycontain a backing layer and a liner that is removed prior to attachmentto the recipient's skin. Transdermal drug delivery patches may also becomprised of a reservoir underlying the backing layer that is separatedfrom the skin of the recipient by a semi-permeable membrane and adhesivelayer. Transdermal drug delivery may occur through passive diffusion,electrotransport, or iontophoresis.

In certain embodiments, a patch contemplated herein may be a slowlydissolving or a time-released patch. In accordance with theseembodiments, a slowly dissolving patch can be an alginate patch. Incertain examples, a patch may contain a detectible indicator dye oragent such as a fluorescent agent. In other embodiments, a tag (e.g.,detectible tag such as a biotin or fluorescently tagged agent) can beassociated with a treatment molecule in order to detect the moleculeafter delivery to the subject. In certain embodiments, one or more oraldelivery patches or other treatment contemplated herein may beadministered to a subject three times daily, twice daily, once a day,every other day, weekly, and the like, depending on the need of thesubject as assessed by a health professional. Patches contemplatedherein may be oral-biodegradable patches or patches for exterior usethat may or may not degrade. Patches contemplated herein may be 1 mm, 2mm, 3 mm, 4 mm to 5 mm in size or more depending on need. In addition,skin patches are contemplated herein for use for example in a subjectsuffering from psoriasis. In treating psoriasis and chronic wounds,Smad7 can be delivered topically using vehicles such as glycerol,carboxymethycellulose. It can also use transdermal system (e.g.,commercially available from 3M) for delivery. Subcutaneous injectioninto the lesion (in normal saline or PBS) can also be used.

In some embodiments, compositions may include short-term, rapid-onset,rapid-offset, controlled release, sustained release, delayed release,and pulsatile release compositions, providing the compositions achieveadministration of a compound as described herein. See Remington'sPharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pa.,1990), herein incorporated by reference in its entirety.

In certain embodiments, the compounds and compositions disclosed hereincan be delivered via a medical device. Such delivery can generally bevia any insertable or implantable medical device, including, but notlimited to stents, catheters, balloon catheters, shunts, or coils. Inone embodiment, the present technology provides medical devices, such asstents, the surface of which is coated with a compound or composition asdescribed herein. The medical device of this technology can be used, forexample, in any application for treating, preventing, or otherwiseaffecting the course of a disease or condition, such as those disclosedherein.

It is contemplated that any molecular biology, cellular biology orbiochemical technique known in the art may be used to generate and/ortest treatments provided herein. In addition, protein chemistrytechniques are contemplated to assess utility of treatments in modelsystems developed herein (e.g., mouse model system).

10. Combination Therapies

It is common in many fields of medicine to treat a disease with multipletherapeutic modalities, often called “combination therapies.” Many ofthe diseases described herein (e.g., inflammatory disease and cancer)are no exception. In some embodiments, to treat inflammatory disordersusing the methods and compositions of the present technology, one wouldcontact a target cell, organ or subject with a Smad7 protein, expressionconstruct or activator and at least one other therapy. These therapieswould be provided in a combined amount effective to achieve a reductionin one or more disease parameter. This process may involve contactingthe cells/subjects with the both agents/therapies at the same time,e.g., using a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell/subject with twodistinct compositions or formulations, at the same time, wherein onecomposition includes the Smad7 agent and the other includes the otheragent.

Alternatively, the Smad7 agent may precede or follow the other treatmentby intervals ranging from minutes to weeks. One would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the therapies would still be able to exert anadvantageously combined effect on the cell/subject. In such instances,it is contemplated that one would contact the cell with both modalitieswithin about 12-24 hours of each other, within about 6-12 hours of eachother, or with a delay time of only about 12 hours. In some situations,it may be desirable to extend the time period for treatmentsignificantly; however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either theSmad7 agent or the other therapy will be desired. Various combinationsmay be employed, where the Smad7 agent is “A,” and the other therapy is“B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are provided. Other agents suitable for use in acombined therapy against an inflammatory disorder include steroids,glucocorticoids, non-steriodal anti-inflammatory drugs (NSAIDS;including COX-1 and COX-2 inhibitors), aspirin, ibuprofen, and naproxen.Analgesics are commonly associated with anti-inflammatory drugs butwhich have no anti-inflammatory effects. An example is paracetamol,called acetaminophen in the U.S. and sold under the brand name ofTylenol. As opposed to NSAIDS, which reduce pain and inflammation byinhibiting COX enzymes, paracetamol has recently been shown to block thereuptake of endocannabinoids, which only reduces pain, likely explainingwhy it has minimal effect on inflammation. A particular agent forcombination use is an anti-TGF-β antibody.

The skilled artisan is directed to Remington's Pharmaceutical Sciences,15th Edition, chapter 33, in particular, pages 624-652, 1990. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating inflammation.

As discussed above, the present technology has particular relevance tothe treatment of DNA damage and/or inflammation resulting from certainanti-cancer therapies, and for the treatment of cancer. Thus, inparticular, the present technology may be applied as a combination withcancer therapies. This process may involve contacting the cells, organ,or patient with the agents/therapies at the same time, including bycontacting the cells, organ or patient with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations at the same time, wherein onecomposition includes the Smad7 agent and the other includes the otheragent. Alternatively, analogous to the chart set forth above, thecompositions can be delivered at different times, including repeateddoses of one or both agents.

Agents or factors suitable for use in a combined therapy include anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, irradiation, microwaves, electronicemissions, and the like. A variety of chemical compounds, also describedas “chemotherapeutic” or “genotoxic agents,” are intended to be of usein the combined treatment methods disclosed herein. In treating canceraccording to the present technology, one would contact the tumor cellswith an agent in addition to the expression construct. This may beachieved by irradiating the localized tumor site; alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition.

Various classes of chemotherapeutic agents are provided for use with incombination with peptides of the present technology. For example,selective estrogen receptor antagonists (“SERMs”), such as Tamoxifen,4-hydroxy Tamoxifen (Afimoxfene), Falsodex, Raloxifene, Bazedoxifene,Clomifene, Femarelle, Lasofoxifene, Ormeloxifene, and Toremifene.

Chemotherapeutic agents contemplated to be of use, include, e.g.,camptothecin, actinomycin-D, mitomycin C. The present technology alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. The agent may beprepared and used as a combined therapeutic composition, or kit, bycombining it with a MUC1 peptide, as described above.

Heat shock protein 90 is a regulatory protein found in many eukaryoticcells. HSP90 inhibitors have been shown to be useful in the treatment ofcancer. Such inhibitors include Geldanamycin,17-(Allylamino)-17-demethoxygeldanamycin, PU-H71 and Rifabutin.

Agents that directly cross-link DNA or form adducts are also envisaged.Agents such as cisplatin, and other DNA alkylating agents may be used.Cisplatin has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for doxorubicin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally. Microtubuleinhibitors, such as taxanes, also are contemplated. These molecules arediterpenes produced by the plants of the genus Taxus, and includepaclitaxel and docetaxel.

Epidermal growth factor receptor inhibitors, such as Iressa, mTOR, themammalian target of rapamycin, also known as FK506-binding protein12-rapamycin associated protein 1 (FRAP1) is a serine/threonine proteinkinase that regulates cell growth, cell proliferation, cell motility,cell survival, protein synthesis, and transcription. Rapamycin andanalogs thereof (“rapalogs”) are therefore provided for use incombination cancer therapy in accordance with the present technology.

Another possible combination therapy with the peptides claimed herein isTNF-α (tumor necrosis factor-alpha), a cytokine involved in systemicinflammation and a member of a group of cytokines that stimulate theacute phase reaction. The primary role of TNF is in the regulation ofimmune cells. TNF is also able to induce apoptotic cell death, to induceinflammation, and to inhibit tumorigenesis and viral replication.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, x-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for x-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to Remington's Pharmaceutical Sciences,15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

In addition to combining Smad7 therapies with chemo- and radiotherapies,it also is contemplated that combination with immunotherapy, hormonetherapy, toxin therapy and surgery. In particular, one may employtargeted therapies such as AVASTIN®, ERBITUX®, GLEEVEC®, HERCEPTIN®, andRITUXAN®.

In other embodiments, to assess the roles and mechanisms of Smad7 withinthe context of oral mucositis, “gene-switch” transgenic mouse modelswere developed to allow control of the level and duration of Smad7transgene expression specifically in oral epithelia. In accordance withthese embodiments, these models may be used to test other genes ordownstream molecules for their effects on oral epithelia and oralmucosa. Thus, these models can be used for, but are not limited to,further analysis of oral wound healing biology and testing therapeuticapproaches to oral wound healing. Molecular Smad7 targets identified inthese studies can provide additional therapeutic targets for subjectssuffering from oral mucositis. Models and resources developed herein canprovide unique tools for analytical studies to identify biomarkers andtherapeutic targets related to Smad7 overexpression and control, forexample, downstream molecules turned on or bound by Smad7 can beidentified as additional therapeutic targets for example, to treat oralmucositis, psoriasis and other conditions aggravated by TGF-β activitiesand NF-κB activities.

D. Kits

In certain embodiments, a kit provided herein may include compositionsdiscussed above for treating a subject having a condition providedherein, such as but not limited to oral mucositis, psoriasis, or woundhealing. The kits can include one or more containers containing thetherapeutic Smad7 compositions of the present technology. Any of thekits will generally include at least one vial, test tube, flask, bottle,syringe or other container, into which compositions may be preferablyand/or suitably aliquoted. Kits herein may also include a kit forassessing biological targets that contribute to a condition providedherein.

E. Methods of Predicting or Evaluating Responses

Also provided are methods for predicting and/or evaluating a response totreatment with Smad7 using by assessing the level of expression of oneor more markers associated with exposure to Smad7. Such markers mayinclude, but are not limited to, Rac1 for cell migration, NF-κB forinflammation, and TGF-β for growth arrest and inflammation. As isdiscussed in the Examples, methods for detection of and/or changes inthe levels of one or more markers associated with Smad7 activity areprovided and/or known in the art. In some embodiments, the level ofexpression of one or more of the Smad7 markers in a subject may beassessed, and based on the level detected, a decision may be made totreat (or to continue or discontinue treatment) with Smad7, or to employan alternate treatment.

The term “detection of” as used herein refers to the ability to measurethe presence or absence of a marker at some repeatable and controlledlevel. Typically, detection is performed over background values, whichmay include the noise (or detection limits) inherent in the testingsystem. As such, there is typically a “lower limit” of detectionassociated with an assay, and in order to be detected, a change may needto be above a certain cut-off level, for example. Determination of suchlimits is well-known in the art.

In some embodiments, detection is performed as compared to controls,which may include, but are not limited to, a comparison with data fromnormal subjects and/or comparable normal tissue (in the same ordifferent subjects) absent the disease or disorder present in thesubject (or the specific tissue of the subject tested). In someembodiments, the comparison may be between levels detected at a varietyof time intervals (and/or locations) in a patient. In some embodiments,the detection needs to be statistically significant as compared tobackground or control levels; the ability to assess significance iswell-known in the art, and exemplified in the Examples.

The term “changes in the levels” as used herein refers to a detectablechange from a control or background level, and or a previously detectedlevel. In some embodiments, the change is an increase as compared toanother level, and in some embodiments the change is a decrease ascompared to another level. In some embodiments, the detectable change(increase or decrease) is statistically significant. In someembodiments, such changes can be assessed quantitatively as at leastabout a 5%, 10%, 25%, 50%, 100%, 200%, 500% or greater change, and/orabout a 5-10%, 10-25%, 10-50%, 25-50%, 50-75%, 50-100%, 100-150%,100-200%, 200-300%, 300-500%, or 500-1000% change.

F. Method of Screening for Additional Biologically Active Fragments

In another aspect, methods of screening for additional biologicallyactive fragments (including, but not limited to truncations) of Smad7are contemplated. In some embodiments, biological activity may beassessed using one of the methods described herein, including thosedescribed below in Examples 5 and 8. Some of the biological activitiesthat can be assessed include, but are not limited to, increasing cellproliferation, reducing or inhibiting cell death, reducing excessiveinflammation, preventing DNA damage, and/or increasing cell migration,as well as animal models treating or preventing one or more disease ordisorders in which such treatment would be helpful as further discussedherein. Such activities can be assessed using one or more assaysincluding, but not limited to, the ability to block phosphorylation ofSmad2 and/or nuclear translocation of the NF-κB p50 subunit, increasecell proliferation, reduce apoptosis and/or radiation-induced DNAdamage, reduce inflammation and/or angiogenesis, promote healing in oralmucositis, surgical wounds, diabetes wounds, and/or wounds associatedwith chronic inflammation in mice and other laboratory models. Somespecific examples include, but are not limited to, immunofluorescence(IF), immunohistochemistry (IHC), and TUNEL assay for apoptosis.

In some embodiments, biologically active fragments are those that areselected to include one or more or all of the activities describedherein. In some embodiments, biologically active fragments selected toinclude only or primarily 1, only or primarily 2, only or primarily 3,only or primarily 4, or only or primarily 5 of the activities describedherein. In some embodiments, biologically active fragments selected toexclude only or primarily 1, only or primarily 2, only or primarily 3,only or primarily 4, or only or primarily 5 of the activities describedherein. In some embodiments, the biologically active fragments areselected to include or to exclude a specific subset of the activitiesdescribed herein. For instance, increased proliferation and migrationmay be sufficient for treating diabetic wounds, whereasanti-inflammation is needed in chronic inflammatory wounds. Reducedapoptosis and DNA damage activities are needed for treating oralmucositis but not for treating surgical wounds

The term “primarily includes” as used herein refers to fragments inwhich although some level of other biological activity may remain, thatactivity is reduced as compared with full-length fragments, whereas theactivity that is considered “primary” remains at about the same or anincreased level as that observed in the full-length native protein.Similarly, the term “primarily excludes” as used herein refers tofragments in which although some level of a particular biologicalactivity may remain, the level of that particular activity is reduced(optionally significantly and/or statistically significantly reduced) ascompared with full-length fragments, whereas one or more otherbiological activities remains at about the same or increased level asthat observed in the full-length native protein.

In some embodiments involving selection of biologically activefragments, the methods include assessing changes in the level ofexpression of one or more biological activities, including increases anddecreases of one or more activities in a selected fragment are assessedas changes in reference to the activities observed in the full-lengthprotein. In some embodiments, one or more biological activities arebeing selected to remain the same as that observed in the full-lengthfragments while other activities may be increased or decreased or eveneliminated (e.g., such fragments would lack one or more of theactivities discussed). In some embodiments, the change is an increase ascompared to another level, and in some embodiments the change is adecrease as compared to another level. In some embodiments, thedetectable change (increase or decrease) is statistically significant.In some embodiments, such changes can be assessed quantitatively as atleast about a 5%, 10%, 25%, 50%, 100%, 200%, 500% or greater change,and/or about a 5-10%, 10-25%, 10-50%, 25-50%, 50-75%, 50-100%, 100-150%,100-200%, 200-300%, 300-500%, or 500-1000% change. In some embodiments,an activity that “remains the same” can still be observed to have somechange from the activity of the full-length protein, but such changemight be limited to, for example, about a 1%, 2%, 5%, 10%, or 20% changeor less.

In a non-limiting example, fragments of interest may include those thatprimarily mediate the anti-inflammatory effect of Smad7. Smad7 peptideshaving this anti-inflammatory function may be sufficient and optionallyan improvement for treating chronic inflammation associated conditions,such as but not limited to, oral mucositis, stomatitis and psoriasis,among others. In another non-limiting example, fragments of interest mayinclude those that primarily mediate cell migration and/or blockingTGF-β-induced growth arrest and/or fibrotic response. Smad7 peptideshaving this cell migration and proliferation function may be sufficient,and optionally an improvement, for enhancing healing that is notassociated with excessive inflammation. Types of wounds that mightbenefit from this form of treatment include, but are not limited to,surgical wounds, fibrotic scarring, and diabetes wounds, defectivehealing and/or scarring among others.

G. Methods of Producing Smad7 Protein

In another aspect, methods for producing Smad7 protein, including any ofthe Smad7 variants, fragments, truncations, fusion proteins (e.g.,PTD-Smad7) described herein are contemplated. The inventors havediscovered methods of producing Smad7 protein at levels and puritysufficient for research, development, or commercialization that includenucleic acid codon optimization. As a result, methods for producingSmad7 including the use of one or more of the codon-optimized Smad7nucleic acid molecules described herein (e.g., within the Examples) areexpressly contemplated.

EXAMPLES

The following examples are included to illustrate various embodiments.It should be appreciated by those of skill in the art that thetechniques disclosed in the examples that follow represent techniquesdiscovered to function well in the practice of the claimed methods,compositions and apparatus. However, those of skill in the art should,in light of the present disclosure, appreciate that many changes may bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe present technology.

Example 1 K5.Smad7 Mice are Resistant to Oral Mucositis

A transgenic mouse model expressing a human Smad7 protein inkeratinocytes (K5.Smad7) was generated as previously described (Han etal., Dev. Cell, 11:301-312, 2006). Transgene expression in oralepithelia was confirmed (FIGS. 7A-B). The mice were bred into in theC57BL/6 background, and 8-10 weeks old male and female transgenic miceand wild-type littermates were used in studies. These mice showedimproved healing of excisional skin wounds (Han et al., Am. J. Pathol.,179:1768-1779, 2011) and radiation-induced oral mucositis.

K5.Smad7 mice and wild-type littermates were exposed to cranialradiation to determine the biological equivalent dose (BED) required toinduce oral mucositis in mice. It was determined that 8 Gy×3 (BED=43.2),a regimen relevant to hypo-fractionated radiotherapy in clinic, was theminimal dose needed to induce oral mucositis (FIGS. 1A-B). To evaluatethe potency of Smad7 effects, they also tested single doses of cranialradiation and found that oral mucositis severity correlated with BEDvalues between 18 Gy (BED=50.4) and 22 Gy (BED=70.4) (FIGS. 1A-B, FIG.7C). By day 9 after initiation of radiation, wild-type mice developedoral ulcers (FIGS. 1A-B).

K5.Smad7 oral mucosa prior to irradiation had morphology similar towild-type mice, but exhibited resistance to radiation-induced oralmucositis (FIGS. 1A-B). Histological analyses revealed that wild-typemice developed oral mucositis (FIG. 1A) similar to that in humans (FIG.1C). The First Affiliated Hospital of Kunming Medical University, Chinaprovided de-identified archived human tissue paraffin sections andapproved the study as an exempt for human subjects. Oral mucositislesions were from the tongue, buccal or oropharyngeal mucosa adjacent torecurrent oral cancers that had undergone radiotherapy. Non-irradiatedoral mucosa sections were from surgically removed sleep apnea oraltissues and a tongue biopsy adjacent to a cyst (mucocele).

K5.Smad7 oral epithelia typically showed radiation dose-dependentdamage, i.e., thinning epithelium and flattened tongue papillae after 8Gy×3 radiation, and more damaged (hypo- or hypertrophic) epithelialcells after 18 Gy and 22 Gy radiation (FIG. 1A). Consistent withincreased leukocyte infiltration in human oral mucositis lesions (FIG.1C), lesions in wild-type mice harbored numerous infiltrated leukocytes(FIGS. 1D-E) consisting of neutrophils, macrophages, and lymphocytes(FIG. 7D); all were substantially reduced in K5.Smad7 oral mucosa (FIGS.1D-E and 7D).

Because it is difficult to capture human oral mucositis pathology at theacute phase, a mouse model was utilized to assess proliferation andapoptosis when ulcers are just formed. Similar to previous reports,proliferative cells were sparse in irradiated wild-type oral epithelium,but were seen more in irradiated K5.Smad7 oral epithelium (FIGS. 1D and1F). Conversely, apoptotic cells were significantly reduced inirradiated K5.Smad7 oral mucosa compared to wild-type mice (FIGS. 1D and1G).

As expected, cells with nuclear NF-κB p50 subunit were significantlyincreased in oral mucositis compared to non-irradiated wild-type oralmucosa (FIGS. 2A-B). Interestingly, TGF-β1, an immune suppressant ininternal organ, but pro-inflammatory in oral mucosa, together with itsactivated signaling mediator, phosphorylated (p) Smad2, were alsoincreased in oral mucositis compared to non-irradiated oral mucosa inwild-type mice (FIGS. 2A-B). Similar changes were also detected in humanoral mucositis lesions (FIGS. 2A-B).

Irradiated K5.Smad7 oral epithelia significantly reduced cells positivefor nuclear NF-κB p50 and pSmad2, even though they still had abundantTGF-β1 protein (FIGS. 2A-B). TGF-β1 mRNA in irradiated wild-type oralmucosa was significantly increased on day 9 and day 10 (FIG. 2C). TGF-β1mRNA level in K5.Smad7 mucosa was similar to wild-type mucosa at earliertime points, but was back to normal by day 10 (FIG. 2C). Although notwishing to be bound by any theory, these data suggest that TGF-β1transcription is not inhibited by Smad7, but its more rapid decline inK5.Smad7 mucosa could be a consequence of accelerated healing.

Phospho-Smad1/5/8, markers for activated BMP signaling, were notaffected by Smad7 before and after radiation (FIG. 7E). This result isconsistent with the ability of Smad7 to preferentially inhibit TGF-βsignaling.

Example 2 Rac1 Contributes to Smad7-Mediated Keratinocyte Migration

To determine if Smad7 contributes to healing in human oralkeratinocytes, Smad7 was knocked down in spontaneously immortalizedhuman oral keratinocytes (NOK-SI) Smad7 knockdown blunted keratinocytemigration after wounding (FIG. 2D and FIG. 8A). Conversely, knockingdown TGF-β1 accelerated keratinocyte migration (FIGS. 8B-8D), consistentwith accelerated wound healing seen in mice null for TGF-β1 or Smad3.

To search for molecular mechanisms associated with Smad7-mediatedkeratinocyte migration, Rac1, a protein indispensable for oral woundhealing was examined Rac1 was reduced after Smad7 knockdown (FIG. 2E).It was expected that TGF-β1 overexpression in oral mucositis wouldactivate Rac1 through a Smad-independent mechanism. However, althoughtotal Rac1 protein increased by 2-fold after irradiation, activated Rac1protein did not change considerably in wild-type tongues (FIG. 2F).

In K5.Smad7 oral mucosa, both total and activated Rac1 weresignificantly increased by 4-fold and 8-fold, respectively, compared towild-type oral mucosa (FIG. 2F). To determine the functionalsignificance of Smad7-induced Rac1 activation, Rac1 was knocked down inprimary keratinocytes isolated from wild-type and Smad7 transgenicneonatal skin, and assays for cell proliferation and migration wereperformed. Rac1 knockdown showed modestly reduced proliferation inwild-type and Smad7 keratinocytes (FIGS. 9A-9C), but almost completelyabrogated Smad7-induced migration (FIG. 2G and FIG. 9D), suggesting thatincreased Rac1 contributes to Smad7-mediated cell migration.

It was observed that increased Rac1 mRNA levels in Smad7 transgenickeratinocytes correlated with total and active Rac1 protein levels(FIGS. 3A-B and FIGS. 10A-B), suggesting that increased Rac1 activationin Smad7 keratinocytes is, at least in part, a consequence of increasedRac1 transcripts. Further, Rac1 protein increased by ˜3-fold (FIG. 3C)after knockdown of individual Smads in NOK-SI cells (FIGS. 10C-10E).These data suggest that normal Smad signaling represses Rac1transcription.

Among the two putative Smad binding elements (SBEs) in the mouse Rac1promoter (−2.1 Kb and -1.5 Kb upstream of the coding sequence), whichare in similar regions of the human Rac1 promoter, chromatinimmunoprecipitation (ChIP) identified Smad-2, -3, -4, and -7 binding tothe -1.5 Kb site (FIG. 3D), but not the -2.1 Kb site in wild-typekeratinocytes; binding of Smad-2, -3 and -4 was significantly reduced inSmad7 transgenic keratinocytes (FIG. 3D).

Luciferase reporter assays using a SBE-containing Rac1-Luc constructshow that knockdown of Smad7 in wild-type keratinocytes significantlyreduced luciferase activity (FIG. 3E). Conversely, Smad7 transgeniccells had increased luciferase activity compared to wild-type cells, andmutating the SBE attenuated this increase (FIG. 3F). Thus, Smad7 bindingto SBE appears necessary to expel signaling Smads to abrogate Rac1repression.

Among known Smad transcriptional co-repressors, it was found that CtBP1bound to the Rac1 promoter SBE-1.5 Kb site in wild-type keratinocytes(FIG. 3G), and Smad7 transgene expression significantly reduced CtBP1binding to the SBE (FIGS. 3G-H). When CtBP1 was knocked down in NOK-SIcells, Rac1 protein and Rac1-Luc activity were increased compared tokeratinocytes transfected with scrambled siRNA (FIGS. 4A-B), suggestingthat CtBP1 binding to SBE-1.5 Kb represses Rac1 expression. Further,knocking down CtBP1 in NOK-SI cells increased their migration (FIG. 4Cand FIG. 10F).

Upon examination of CtBP1 protein in radiation-induced oral mucositis,it was found that CtBP1 is barely detectable in non-irradiated mouse andhuman oral mucosa (FIGS. 4D-4F); however, CtBP 1 positive cells weresignificantly increased in irradiated oral mucosa of wild-type andK5.Smad7 mice as well as in human oral mucositis (FIGS. 4D-4F).Additionally, CtBP1 mRNA in irradiated wild-type oral mucosa wassignificantly increased on day 9 and day 10 (FIG. 4G). CtBP1 mRNA levelin K5.Smad7 mucosa was similar to wild-type mucosa at earlier timepoints, but declined to normal by day 10 (FIG. 4G). These resultsindicate that Smad7 does not reduce CtBP1 mRNA but instead inhibitsCtBP1 binding to the Rac1 promoter by repelling the Smad/CtBP1 complexfrom the SBE binding site; further, more rapid CtBP1 reduction inK5.Smad7 mucosa serves as a marker of healing.

Example 3 Tat-Smad7 Alleviates Radiation-Induced Oral Mucositis

Smad7 transgene's ability to block multiple pathological processes oforal mucositis prompted us to explore if localized Smad7 delivery can beused to prevent and treat oral mucositis. Because Smad7 is a nuclearprotein, local Smad7 delivery needs to allow Smad7 to rapidly enter intocells before saliva washes off the protein. Thus, a recombinant humanSmad7 with an N-terminal Tat-tag allowing proteins to rapidly permeatethe cell membrane and enter the nucleus was produced. A V5 epitope wasadded to the C-terminal end of the Tat-Smad7 protein to track Tat-Smad7cell penetration (FIGS. 11A-11D).

Using its ability to block Smad2 phosphorylation, Tat-Smad7 bioactivitywas tested (FIG. 11C). Tat-Cre recombinant protein with the same tags asa control (FIGS. 11E-F) was produced, and cloned into the pET101-Topoprotein expression vector (Invitrogen) that contains a sequence encodingC-terminal 6×His (SEQ ID NO: 40). Tat-Cre was transformed into BL-21STAR™ E. coli (Invitrogen) to produce Tat-Cre protein and was purifiedwith Ni-NTA column.

The purity and size of both proteins was verified using SDS-PAGEelectrophoresis. To evaluate transduction and activity of Tat-Smad7protein in vitro, Tat-Smad7 was added to primary mouse keratinocytes.Slides were fixed in cold methanol for 5 minutes and stained for V5 andpSmad2. Tat-Cre activity was verified by digesting a 1,460 bp floxedfragment from the 7,650 bp vector pLL3.7 (Addgene). For in vivotreatments, 30 μL 50% glycerol/PBS as a vehicle control and Tat-Cre as anon-irrelevant protein control were used. Tat-Smad7 or Tat-Cre (in 30 μL50% glycerol/PBS, doses and regimens are specified in each figure) wastopically applied to mouse oral cavity and mice were restricted fromoral intake for 1 hour.

For oral mucositis prevention, both Tat-Smad7 and Tat-Cre (in 50%glycerol/PBS) were topically applied to the oral cavity of 8-10 week oldC3H females (Jackson Laboratory) or C57BL/6 mice daily, starting 24hours prior to radiation through day 8 after initiation of radiation.Treated tissues were examined on day 9. Mouse tongues were harvested,fixed in 10% formalin, embedded in paraffin, and cut into 5 μm sections.Histological changes were analyzed and ulcers were measured using H&Estained slides. An additional group received Palifermin treatment with aclinical regimen, i.e., 6.25 mg kg⁻¹ (i.p.) daily for 3 days prior toirradiation, and daily for 3 days 24 hours after the last dose ofradiation.

Tat-Cre showed no effect compared to vehicle controls (FIGS. 5A-B).Tat-Smad7 treatments showed preventive effects on ulcer formationsimilar to Palifermin (FIG. 5A). The dose-dependent effect of Tat-Smad7was more obvious when used on animals given a 20 Gy (BED=60) single doseof radiation that induced larger oral ulcers than fractionated radiation(FIG. 11G). Microscopically, both Palifermin and Tat-Smad7 treated oralmucosa prevented open ulceration in the majority of cases (FIG. 5B).Palifermin-treated mucosa exhibited more keratinocyte down-growth butalso more damaged keratinocytes (condensed or charcoal-like nuclei,swelled mono- or multi-nucleated cells and shattered nuclear fragmentsin confide layers) than Tat-Smad7-treated mucosa (FIG. 5B).Immunostaining revealed that Palifermin increased proliferation moresignificantly than Tat-Smad7. Tat-Smad7 reduced apoptosis, leukocyteinfiltration, nuclear pSmad2 and NF-κB p50, but Palifermin did not(FIGS. 5B-5G).

To test whether Tat-Smad7 can be used to treat existing oral mucositis,mice were exposed to fractionated (8 Gy×3) cranial radiation andTat-Smad7 (topically) or Palifermin (6.25 mg kg⁻¹, i.p.) was applieddaily from day 6 after initiation of radiation (when mucosal damage wasobvious) till day 9. Treated tissues were examined on day 10. Althoughbeginning post-radiation administration of Palifermin at earlier timepoints than the current protocol reduced oral mucositis in mice,Palifermin administration with the current protocol did not accelerateulcer closure (FIG. 6A), regardless of its hyperproliferative effect onthe entire oral mucosa (FIG. 6B). This is not surprising, as Paliferminis approved to prevent but not treat oral mucositis.

Tat-Smad7 treated oral mucositis reduced ulcer sizes and pathologicalalterations after both fractionated and single dose radiation (FIGS.6A-B and FIGS. 12A-12G). Away from ulcers, Tat-Smad7 treated oral mucosaexhibited less hyperplasia and more differentiated epithelia thanPalifermin-treated oral mucosa (FIG. 6B). With a 20 Gy single doseradiation that caused slower healing than fractionated radiation, theeffect of Tat-Smad7 on recovery after wound closure was more obvious.When vehicle treated ulcer was just re-epithelialized, Tat-Smad7 treatedmucosa had almost recovered to normal morphology (FIG. 6C).

Consistent with observations in K5.Smad7 mice, Tat-Smad7 increased Rac1promoter activity and reduced CtBP1 binding to the SBE of the mouse Rac1promoter (FIGS. 12G and 12I), and increased Rac1 protein in mouse oralmucositis and human oral keratinocytes (FIGS. 6D-E).

Tat-Smad7-treated human oral keratinocytes after wound scratch hadaccelerated wound closure (FIG. 6F and FIG. 13A). Further, irradiatedhuman oral keratinocytes increased nuclear pSmad2 and NF-κB p50, whichwere attenuated by Tat-Smad7 treatment (FIG. 13B). In contrast, althoughTat-Smad7 penetrated oral cancer cells efficiently (FIG. 13C), it didnot further elevate Rac1 protein level that is already abundant incancer cells (FIG. 13D). This result could account for faster migrationof cancer cells than normal keratinocytes (FIG. 6F and FIGS. 13A,13E-13H), and the lack of an effect of Tat-Smad7 on migration in twooral cancer cell lines: MSK921 which does not contain genetic loss ofTGF-β signaling components; and Ca127 which has a mutated Smad4 (FIGS.13E-13H).

Colony assays show that survival of human oral keratinocytes wasslightly increased by Tat-Smad7 treatment with or without radiation(FIG. 6G). Consistent with the notion that reduced survival afterirradiation is more prominent in cancer cells than in normal cells, SCCcells showed substantial reductions in cell survival after radiation.Treatment with Tat-Smad7 did not affect survival in SCC cells with orwithout radiation (FIG. 6G).

Example 4 Design of a Cell-Penetrating Smad7 Protein

It was hypothesized that in order to be effective as a therapeutic,SMAD7 needed to be able to penetrate cells efficiently. In order toachieve this, the Smad7 sequence was modified to include a proteintransduction domain.

The Tat sequence from HIV was selected to test with Smad7 as a proteintransduction domain. The nucleotide and protein sequences of Tat thatwere used in fusion proteins with Smad7 and Smad7 fragments are derivedfrom Cardarelli et al., Traffic Apr 9(4):528-39 (2008). The Tatnucleotide and amino acid sequences are provided below:

ggccgtaaaaaacgccgtcaacgccgccgt (SEQ ID NO: 1) G R K K R R Q R R R(SEQ ID NO: 2)Fusion proteins were prepared having Tat directly linked in frame tohuman Smad7 complementary DNA (cDNA) either at the 5′ or 3′ ends ofSmad7 as shown below:

5′ Tat: Ggccgtaaaaaacgccgtcaacgccgccgt(SEQ ID NO: 7)-Smad7 3′ Tat:Smad7-Ggccgtaaaaaacgccgtcaacgccgccgt(SEQ ID NO: 8)The 5′ Tat-Smad7 construct included a 3′ V5 tag sequence, and was clonedinto the pGEX-6p-1 protein expression vector (New England Biolabs) tomake a GST-Tat-Smad7 fusion protein. Tat-Smad7 gene was transformed intoBL-21 Star Escherichia coli (Invitrogen) to produce Tat-Smad7 protein.The protein was purified by glutathione column purification and elution,using enzymatic cleavage from the Glutathione S Transferase (GST) fusion(Precision enzyme, GE Life Sciences).

While creating a PTD-Smad7 fusion protein, a V5 tag at the 3′ end wasincluded to monitor Tat-Smad7 penetration into cells by immunostainingusing a V5 antibody. This epitope tag can be deleted for use in theclinic (e.g., by re-cloning the sequence in the absence of the V5 tag),if appropriate.

A PTD-Smad7 fusion protein (Tat-Smad7-V5-6H) (“6H” disclosed as SEQ IDNO: 40) was also created having a 6-Histidine (6-H) tag (SEQ ID NO: 40)for protein purification, and is shown below. Tat-Smad7-V5-6H (“6H”disclosed as SEQ ID NO: 40) has the following nucleotide sequence: 1-53include the 5′ sequence of pET-TOPO; 54-1365 include Tat-Smad7;1366-1497 include 3′ pET-TOPO containing the V5 epitope and 6×His tag(SEQ ID NO: 40) (V5 includes 1393-1434, His tag includes 1444-1461, andthe Stop includes 1462-1464).

Tat-human Smad7, codon-optimized for protein production, cloned topET101/D-Topo vector is shown below:

(SEQ ID NO: 9) ttcccctctagaaataattttgtttaactttaagaaggaattcaggagcccttcaccatg (SEQ ID NO: 10) Mcgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgctcggc cctggtccgtR K K R R Q R R R G F R T K R S A L V Rcgcctgtggcgctcccgtgctccgggtggtgaagatgaagaagaaggtgc tggcggcggtR L W R S R A P G G E D E E E G A G G Gggcggtggcggtgaactgcgtggcgagggtgcaaccgatagtcgtgcaca cggtgcaggcG G G G E L R G E G A T D S R A H G A Gggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcgg cgcgaaaggtG G G P G R A G C C L G K A V R G A K Gcatcaccatccgcacccgccggcagcaggtgcaggtgcagctggcggtgc ggaagccgatH H H P H P P A A G A G A A G G A E A Dctgaaagccctgacccatagtgtcctgaaaaaactgaaagaacgtcagct ggagctgctgL K A L T H S V L K K L K E R Q L E L Lctgcaagcagtagaatcccgtggcggtacccgtacggcttgtctgctgct gccgggtcgtL Q A V E S R G G T R T A C L L L P G Rctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcaca accgccgagcL D C R L G P G A P A G A Q P A Q P P Stcttacagcctgccgctgctgctgtgtaaagtgtttcgttggccggacct gcgccacagtS Y S L P L L L C K V F R W P D L R H Stccgaagttaaacgcctgtgctgttgcgagagctatggcaaaattaaccc ggaactggttS E V K R L C C C E S Y G K I N P E L Vtgttgcaatccgcaccatctgtctcgtctgtgtgaactggagagcccgcc gccgccgtatC C N P H H L S R L C E L E S P P P P Ytctcgttacccgatggatttcctgaaaccgactgcagattgcccggacgc agtcccgtcaS R Y P M D F L K P T A D C P D A V P Stcggctgagaccggcggcaccaactatctggcaccgggcggtctgagtga ttcccagctgS A E T G G T N Y L A P G G L S D S Q Lctgctggaaccgggcgaccgttcacattggtgtgtggttgcctattggga agagaaaacgL L E P G D R S H W C V V A Y W E E K Tcgtgtcggtcgcctgtactgcgtacaggaaccgtcgctggatatctttta tgacctgccgR V G R L Y C V Q E P S L D I F Y D L Pcagggcaatggtttctgtctgggccaactgaactcagataataaatcgca gctggtgcaaQ G N G F C L G Q L N S D N K S Q L V Qaaagttcgctcaaaaattggctgcggtatccagctgacccgtgaagttga cggtgtctggK V R S K I G C G I Q L T R E V D G V Wgtatataaccgcagctcttacccgatttttatcaaaagtgccaccctgga taatccggacV Y N R S S Y P I F I K S A T L D N P Dtcccgtacgctgctggtccacaaagtatttccgggcttctcaatcaaagc gttcgattacS R T L L V H K V F P G F S I K A F D Ygagaaagcctactcgctgcagcgcccgaacgaccatgaattcatgcagca accgtggacgE K A Y S L Q R P N D H E F M Q Q P W Tggttttactgtgcagatctctttcgttaaaggctggggtcaatgctacac ccgtcagtttG F T V Q I S F V K G W G Q C Y T R Q Fatctcgtcctgtccgtgctggctggaagtgattttcaatagccgcaaggg cgagctcaatI S S C P C W L E V I F N S R K G E L Ntcgaagcttgaaggtaagcctatccctaaccctctcctcggtctcgattc tacgcgtaccS K L E G K P I P N P L L G L D S T R Tggtcatcatcaccatcaccattgagtttgatccggctgctaacaaagccc gaaagga G H H H H H H

A comparison of the protein sequence of Tat-Smad7-v5 and Smad7 isprovided below. The first amino acid of Smad7 in Tat-Smad7 is not M(unlike Smad7), because Tat-Smad7 is designed to be in-frame with Tatand/or GST to form a GST fusion protein. Tat-Smad7 is then cleaved fromthe GST fusion protein after purification. Upper case nucleotidesidentify the V5 tag. Underlined italics indicate amino acids from theoptional pET101-Topo backbone vector.

Below a Tat-Smad7-v5 and Smad7 comparison is presented:

Tat-Smad7-V5   1 gsgrkkrrqrrrgfrtkrsalvrrlwrsrapggedeeegagggggggelrhuman Smad7   1 ------------mfrtkrsalvrrlwrsrapggedeeegagggggggelrTat-Smad7-V5  51 gegatdsrahgaggggpgragcclgkavrgakghhhphppaagagaaggahuman Smad7  39 gegatdsrahgaggggpgragcclgkavrgakghhhphppaagagaaggaTat-Smad7-V5 101 eadlkalthsvlkklkerqlelllqavesrggtrtaclllpgrldcrlgphuman Smad7  89 eadlkalthsvlkklkerqlelllqavesrggtrtaclllpgrldcrlgpTat-Smad7-V5 151 gapagaqpaqppssyslplllckvfrwpdlrhssevkrleccesygkinphuman Smad7 139 gapagaqpaqppssyslplllckvfrwpdlrhssevlalcccesygkinpTat-Smad7-V5 201 elvccnphhlsrlcelesppppysrypmdflkptadcpdavpssaetggthuman Smad7 189 elvccnphhlsrlcelesppppysrypmdflkptadcpdavpssaetggtTat-Smad7-V5 251 nylapgglsdsqlllepgdrshwcvvayweektrvgrlycvqepsldifyhuman Smad7 239 nylapgglsdsqlllepgdrshwcvvayweektrvgrlycvqepsldifyTat-Smad7-V5 301 dlpqgngfclgqlnsdnksqlvqkvrskigcgiqltrevdgvwvynrssyhuman Smad7 289 dlpqgngfclgqlnsdnksqlvqkvrskigcgiqltrevdgvwvynrssyTat-Smad7-V5 351 pifiksatldnpdsrtllvhkvfpgfsikafdyekayslqrpndhefmqqhuman Smad7 339 pifiksatldnpdsrtllvhkvfpgfsikafdyekayslqrpndhefmqqTat-Smad7-V5 401 pwtgftvqisfvkgwgqcytrqfisscpcwlevifnsrk gelnskle GKPhuman Smad7 389 pwtgftvqisfvkgwgqcytrqfisscpcwlevifnsr------------Tat-Smad7-V5 451 IPNPLLGLDST (SEQ ID NO: 11) human Smad7 427----------- (SEQ ID NO: 12)

Example 5 Additional Assays for PTD-Smad7 Protein Activity

Immunofluorescence (IF), Immunohistochemistry (IHC), and TUNEL Assay forApoptosis.

IF and IHC were performed as previously described (Han, G., Li, F., TenDijke, P. & Wang, X. J. Temporal smad7 transgene induction in mouseepidermis accelerates skin wound healing. Am J Pathol 179, 1768-1779(2011)). Primary antibodies used were guinea pig antibody to K14 (1:400,Fitzgerald, 20R-CP200), rat antibody to CD4 (1:20, BD Bioscience,550278), Ly-6G (1:20, BD Bioscience, 550291), BM8 (antibody to F4/80,1:20, Invitrogen, MF48000), FITC-labeled antibody to BrdU (BDBioscience, 347583), rat antibody to CD45 (1:50, BD Bioscience, 550539)for mouse samples, mouse antibody to CD45 (1:50, Abcam, Ab781) for humansamples, chicken antibody to TGF-β1 (1:50, R&D, AF-101-NA), rabbitantibody to CtBP1 (1:100, Millipore, 07-306), rabbit antibody to NF-κBp50 (1:200, Santa Cruz Biotechnology, SC-7178), rabbit antibody to PCNA(1:200, Santa Cruz Biotechnology, SC-7907), rabbit antibody to pSmad2(1:100, Cell Signaling Technology, 3101), and mouse antibody to V5(1:500, Invitrogen, 460705). For IF, secondary antibodies to differentspecies IgG were Alexa Fluor® 594 (red) or 488 (green) conjugated (1:200for all, Invitrogen). For IHC, secondary biotinylated antibodies todifferent species IgG (1:300, Vector Labs) were used and were developedusing Vectastain ABC kit (Vector Labs). A Terminal deoxynucleotidyltransferase uridine nick end-labeling (TUNEL, G3250) kit (Promega) wasused on formalin fixed tissue sections to detect apoptotic cells. BrdUlabeling was performed in vivo by i.p. injection of 0.125 mg g⁻¹ BrdU 1hour prior to euthanization. PCNA or BrdU were quantified as cells mm⁻¹epithelial length including all epithelial cells, TUNEL or CD45-positivecells as cells mm⁻¹ epithelial length including all epithelial layersand stroma above the muscle layer, nuclear pSmad2 or NF-κB p50 positivecells as the number of positive cells/existing total remainingepithelial cells (i.e., excluding sloughed epithelial cells induced byirradiation). Consecutive fields of slides were used to countBrdU-labeled cells using MetaMorph software.

Cell Culture.

Smad7 transgenic and wild-type primary keratinocytes were prepared fromneonatal mouse skin as previously described (Han, G., Li, F., Ten Dijke,P. & Wang, X. J. Temporal smad7 transgene induction in mouse epidermisaccelerates skin wound healing. Am J Pathol 179, 1768-1779 (2011)), andcultured in PCT medium (CELLnTEC). Spontaneously immortalized normaloral keratinocytes (NOK-SI) derived from gingival tissues of healthyvolunteers were cultured and maintained in defined keratinocyte medium(Castilho, R. M., et al. Rac1 is required for epithelial stem cellfunction during dermal and oral mucosal wound healing but not for tissuehomeostasis in mice. PloS one 5, e10503 (2010)). Oral cancer cells Ca127(ATCC) and MSK921 were cultured (D. Raben's lab, fingerprinted byUniversity of Colorado Cancer Center Tissue Culture Core) in DulbeccoModified Eagle Medium supplemented with 10% fetal bovine serum (GIBCO®;Invitrogen). To assess the effect of Tat-Smad7 in irradiated cells, theabove human cell lines were cultured in chamber slides (BD Bioscience,354108), irradiated with 3 Gy, and Tat-Smad7 (1 μg mL⁻¹) was added tothe culture medium immediately after irradiation. Cells were fixed in100% cold methanol 4 hours after Tat-Smad7 treatment for immunostainingof pSmad2, NF-κB p50 and V5.

Transfection with siRNA.

When cultured keratinocytes reached 70% confluency, 100 nM of targetsiRNA or scrambled siRNA (Dharmacon) was transfected usingLIPOFECTAMINE® 2000 (Invitrogen). Cells were harvested 48-72 hours aftertransfection and subjected to western analyses to determine knockdownefficiency. For migration assays, siRNA was transfected when cells wereplated. Target siRNAs included in this study are: mouse siRac1-1(Invitrogen, MSS237708) and siRac1-2 (IDT, MMC.RNAI.N009007.12.3); humansiSmad2 (Dharmacon, L-003561-00-0005), siSmad3 (Invitrogen, HSS106252),and siSmad4 (Invitrogen, HSS118066); human siCtBP1-1 and siCtBP1-2;human siSmad7-1 and siSmad7-2; human TGF-β1 (Dharmacon,J-012562-08-0005); mouse siSmad7.

In Vitro Keratinocyte Proliferation Assay.

In vitro keratinocyte proliferation was determined by BrdU incorporationin wild-type and Smad7 transgenic keratinocytes. Cells at 70% confluencywere transfected with Rac1 siRNAs, and changed to regular culture medium24 hours later. An in situ cell proliferation kit (Roche AppliedScience) was used to perform in vitro BrdU labeling and detection, andMetaMorph software was used to count BrdU-labeled cells.

In vitro cell migration assays. When cells reached 100% confluency, thecells were treated with mitomycin C (Sigma) at 10 μg mL⁻¹ for 2 hours toinhibit cell proliferation and a scratch wound was introduced with aFisherbrand pipet tip. Cell migration was photographed daily. Migrationassays were performed when cells reached confluency after 24 to 36 hoursof siRNA transfection, and Image-J software was used to document cellmigration as the wound area occupied with migrating cells. For Tat-Smad7treatment, cells were exposed to Tat-Smad7 protein at 1 μg mL⁻¹ orvehicle control (PBS) in medium after wound scratch, and medium waschanged every other day with freshly added Tat-Smad7 until migratingcells fully covered the scratched wound.

Cell Survival Assay.

Cell survival assays were performed as previously described (Munshi, A.,Hobbs, M. & Meyn, R. E. Clonogenic cell survival assay. Methods inmolecular medicine 110, 21-28 (2005)), with slight modifications.Briefly, cells were plated in 12-well plates at 500 cells well⁻¹ fornon-irradiated wells, and increased up to 1,500 cells well⁻¹ along withincreased radiation doses. Cells were irradiated 24 hours after theywere plated. Tat-Smad7 was added at 1 μg mL⁻¹ or the same volume of PBSused to dissolve Tat-Smad7 (control) to culture medium of irradiated andnon-irradiated cells. The medium was changed every other day withfreshly added Tat-Smad7 or PBS for 10 to 14 days. Colonies were fixed inmethanol, stained in 0.5% crystal violet solution (containing 25%methanol), counted and the average from 4 wells in each experiment wascalculated. Two to three separate experiments were performed for eachcell line. The relative surviving fraction was calculated as previouslydescribed, i.e., the absolute surviving fraction (colony numbers/totalplated cells) under each radiation dose divided by the absolutesurviving fraction of non-irradiated cells.

Western Analysis.

Protein extraction and western analyses were performed as previouslydescribed (Li, A. G., Lu, S. L., Zhang, M. X., Deng, C. & Wang, X. J.Smad3 knockout mice exhibit a resistance to skin chemicalcarcinogenesis. Cancer Res 64, 7836-7845 (2004)). The antibodies used inthis study included rabbit antibody to Smad7 (1:500), rabbit antibodiesto Smad2 (1:300, Zymed, 51-1300) and Smad4 (1:300, Epitomics, 1676-1),rabbit antibody to Smad3 (1:300, Cell Signaling Technology, 9513), mouseantibody to Rac1 (1:500, BD Biosciences, 610651), rabbit antibody toCtBP1 (1:500, Millipore, 07-306), mouse antibody to tubulin (1:3000,Sigma, T5168), mouse antibody to GAPDH (1:5000, Abcam, Ab8245) and goatantibody to actin (1:1000, Santa Cruz Biotechnology, SC1616). Gray-scaleimages were obtained using the ODYSSEY® v.1.2 software (LI-CORBiosciences).

Rac1 Activation Assay.

Active GTP-bound Rac1 was examined using a BIOCHEM™ Kit for Rac1activation (Cytoskeleton Inc, BK035). Wild-type and Smad7 transgenickeratinocytes were cultured in 15 cm diameter tissue culture plates andprepared protein lysates using the provided lysis buffer. To assay Rac1activity, 1 mg of cell lysate was used. To examine total Rac1 and Smad7proteins, 50 μg of lysate was used. To measure GTP-bound Rac1 in mousetongues, half of the tongue was ground to a powder in liquid nitrogenand lysed with lysis buffer to extract protein, GTP-bound Rac1 wasassayed in 2 mg of protein lysate per sample and 50 μg of protein lysatewas loaded for total Rac1 protein western blot.

ChIP Assays.

ChIP assays were performed using the ChIP-IT express kit (Active Motive,53009) as previously described (Hoot, K. E., et al. HGF upregulationcontributes to angiogenesis in mice with keratinocyte-specific Smad2deletion. J Clin Invest 120, 3606-3616 (2010); Hoot, K. E., et al.Keratinocyte-specific Smad2 ablation results in increasedepithelial-mesenchymal transition during skin cancer formation andprogression. Owens, et al., J. Clin. Invest 118, 2722-2732 (2008).Smad4-dependent desmoglein-4 expression contributes to hair follicleintegrity. Owens, et al., Dev. Biol. 322:156-166 (2008). DNA-proteincomplex was isolated from primary mouse keratinocytes. For ChIP, 6.3 μgsheared chromatin was incubated with protein-G magnetic beads and 2 μgeach of rabbit antibodies to Smad2 (Cell Signaling Technology, 3122),Smad3 (Cell Signaling Technology, 9523), Smad4 (Cell SignalingTechnology, 9515), Smad7 antibody (Santa Cruz Biotechnology, SC-11392),CtBP1 (Millipore) or a negative control rabbit IgG (Santa CruzBiotechnology, SC-2027). Eluted DNA from the protein-DNA complex wasused for PCR analyses, and CtBP 1 binding to the Rac1 promoter wascompared in wild-type and Smad7 transgenic keratinocytes by ChIP bandintensities on gel images or by quantitative PCR using Power SYBR GreenMaster Mix (Applied Biosystems). Primers used to amplify the Rac1SBE-1.5 Kb promoter regions:

(SEQ ID NO: 13) 5′-TGGAATTCCTGGTCTGGTTT-3′ (sense) (SEQ ID NO: 14)5′-GCCAAGCTGCTCTTCCAGTA-3′ (antisense) (SEQ ID NO: 15)5′-TCTCAGGGGGCCAAAGGTGTT-3′ (sense) (SEQ ID NO: 16)5′-TCCCAGCACCTGAATCACATGG-3′ (antisense)

Rac1 Promoter Luciferase Reporter Construct, Site-Directed Mutagenesisand Luciferase Assay.

The 883 bp fragment of −1671 bp to −789 bp of the Rac1 promoter,encompassing the SBE-1.5 Kb site, was amplified from wild-type mouse DNAusing 5′ XhoI and 3′ HindIII tagged primers, and this Rac1 promoterfragment was cloned into pGL4.26 vector (Promega) to make the Rac1promoter-pGL4.26 luciferase reporter (Rac1-Luc) construct. Forsite-directed mutagenesis, the SBE sequence 5′-TGTCTGTGCT-3′ (SEQ ID NO:17) was mutated to 5′-TGATAGAGCT-3′ (SEQ ID NO: 18). Rac1-Luc andpGL4.74 (1:20) were co-transfected with Smad7 siRNA, CtBP1 siRNA orscrambled siRNA using Lipofectamine 2000 (Invitrogen) to primary mousekeratinocytes, or primary mouse keratinocytes with Tat-Smad7 treatment(1 μg mL⁻¹). Cell lysates were collected and luciferase assays wereperformed 48 hours after transfection or Tat-Smad7 treatment, using theDUAL-LUCIFERASE® Reporter Assay kit (Promega) following manufacturer'sinstructions. Rac1-luciferase activity was measured with the Glomaxmachine (Promega) and expressed by the ratio of firefly activity toRenilla activity. Primers used for amplification of Rac1 promotersequence were:

5′-ATCCTCGAG-TATCCTCCAGGTCTGGG-3′ (SEQ ID NO: 19)5′-GCCAAGCTT-AGCGTCCAGCGTTAACCTG-3′ (SEQ ID NO: 20)

Statistical Analysis.

Statistical differences in molecular analyses and oral mucositis ulcersize were analyzed using the Student's t-test and all data was presentedby mean±s.d. except ulcer size, which was presented by mean±s.e.m. Oralmucositis incidences were analyzed by Fisher's exact test.

Example 6 Codon Optimization for Smad7 Protein Production in E. coli orYeast

Although many mammalian proteins can be produced in bacteria withoutnucleotide sequence modification, the analysis indicated that the Smad7nucleotide sequence would need to be modified to allow proteinexpression in bacteria.

Analysis of Smad7 cDNA mammalian codon use revealed nine arginine aminoacids coded for by the following nucleotides: 7-9, 43-45, 169-171,403-405, 490-492, 526-528, 526-528, 823-825, 1057-1059 are a rare codon(AGG, codon utilization 1.7%). Since these codons are rare codons inbacteria, it is expected that they could halt or reduce proteintranslation and/or production in bacteria. The amino acids coded for byrare arginine codons are indicated by bold capitals below in theillustrated human Smad7 protein, including arginines at positions 3, 15,57, 135, 164, 169, 176, 275, and 353. Additionally, the followingarginine codons also have low frequency usages. CGA (3.5% codonutilization): nucleotides 16-18, 136-138, 199-201, 598-600, which codefor arginine at positions 6, 46, 67, 200; CGG (5.4% codon utilization):nucleotides 31-33, 112-114, 316-318, 772-774, 940-942, 973-975,1135-1137, 1276-1278, which code for arginine at positions 11, 38, 106,258, 314, 325, 379, 426; AGA (2.8% codon utilization): nucleotides637-639, 814-816, which code for arginine at positions 213, 272. Thesearginine residues are highlighted in bold upper case R below and theyare changed to CGC in at least one of the codon-optimized nucleic acidsequences (20.6% codon utilization):

(SEQ ID NO: 12)   1mfRtkRsalv RrlwRsrapg gedeeegagg gggggelRge gatdsRahga  51ggggpgRagc clgkavrgak ghhhphppaa gagaaggaea dlkalthsvl 101kklkeRqlel llqavesrgg trtaclllpg rldcRlgpga pagaqpaqpp 151ssyslplllc kvfRwpdlRh ssevkRlccc esygkinpel vccnphhlsR 201lcelespppp ysRypmdflk ptadcpdavp ssaetggtny lapgglsdsq 251lllepgdRsh wcvvayweek tRvgRlycvq epsldifydl pqgngfclgq 301lnsdnksqlv qkvRskigcg iqltRevdgv wvynrssypi fiksatldnp 351dsRtllvhkv fpgfsikafd yekayslqRp ndhefmqqpw tgftvgisfv 401kgwgqcytrq fisscpcwle vifnsR

Based on this analysis, it was decided to optimize the Smad7 nucleotidesequence to codons that were believed to allow increased Tat-Smad7protein production in E. coli or yeast. Provided below is the optimizednucleic acid codon sequence made by Genscript. Briefly, the sequence hasthe following composition: nucleotides 1-6 include the restrictionrecognition site for BamHI; nucleotides 7-36 include the Tat sequence;nucleotides 37-1314 include codon-optimized human Smad7 cDNA;nucleotides 1342-1383 include the V5 epitope; nucleotides 1384-1386 arethe stop codon; and nucleotides 1387-1392 including the restrictionrecognition site for SalI. In this sequence, ATG is removed to be usedwith GST. The entire designed sequence was converted to E. coli codonsbased on “Codon-Usage Database.” The initial optimized Smad7 sequence(SEQ ID NO: 23) is shown below:

(SEQ ID NO: 23)    1ggatccggcc gtaaaaaacg ccgtcaacgc cgccgtggtt tccgtacgaa acgctcggcc   61ctggtccgtc gcctgtggcg ctcccgtgct ccgggtggtg aagatgaaga agaaggtgct  121ggcggcggtg gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag tcgtgcacac  181ggtgcaggcg gtggcggtcc gggtcgtgct ggttgctgtc tgggtaaagc tgtgcgcggc  241gcgaaaggtc atcaccatcc gcacccgccg gcagcaggtg caggtgcagc tggcggtgcg  301gaagccgatc tgaaagccct gacccatagt gtcctgaaaa aactgaaaga acgtcagctg  361gagctgctgc tgcaagcagt agaatcccgt ggcggtaccc gtacggcttg tctgctgctg  421ccgggtcgtc tggattgccg tctgggtccg ggtgcaccgg ctggtgcgca gccggcacaa  481ccgccgagct cttacagcct gccgctgctg ctgtgtaaag tgtttcgttg gccggacctg  541cgccacagtt ccgaagttaa acgcctgtgc tgttgcgaga gctatggcaa aattaacccg  601gaactggttt gttgcaatcc gcaccatctg tctcgtctgt gtgaactgga gagcccgccg  661ccgccgtatt ctcgttaccc gatggatttc ctgaaaccga ctgcagattg cccggacgca  721gtcccgtcat cggctgagac cggcggcacc aactatctgg caccgggcgg tctgagtgat  781tcccagctgc tgctggaacc gggcgaccgt tcacattggt gtgtggttgc ctattgggaa  841gagaaaacgc gtgtcggtcg cctgtactgc gtacaggaac cgtcgctgga tatcttttat  901gacctgccgc agggcaatgg tttctgtctg ggccaactga actcagataa taaatcgcag  961ctggtgcaaa aagttcgctc aaaaattggc tgcggtatcc agctgacccg tgaagttgac 1021ggtgtctggg tatataaccg cagctcttac ccgattttta tcaaaagtgc caccctggat 1081aatccggact cccgtacgct gctggtccac aaagtatttc cgggcttctc aatcaaagcg 1141ttcgattacg agaaagccta ctcgctgcag cgcccgaacg accatgaatt catgcagcaa 1201ccgtggacgg gttttactgt gcagatctct ttcgttaaag gctggggtca atgctacacc 1261cgtcagttta tctcgtcctg tccgtgctgg ctggaagtga ttttcaatag ccgcaagggc 1321gagctcaatt cgaagcttga aggtaagcct atccctaacc ctctcctcgg tctcgattct 1381acgtgagtcg ac

A nucleotide sequence comparison between Tat-Smad7-V5 (SEQ ID NO: 23)and human Smad7 (SEQ ID NO: 22) cDNA is provided below. Human Smad7 andcodon-optimized Tat-Smad7-V5 share 68% codon homology. Human Smad7 andcodon-optimized Tat-Smad7 share 71% codon homology. Human Smad7 andcodon-optimized Smad7 share 73% codon homology.

Alignment: Global DNA alignment against reference moleculeParameters: Scoring matrix: Linear (Mismatch 2, OpenGap 4, ExtGap 1)Reference molecule: human Smad7 mRNA, Region 1-1281Number of sequences to align: 2Settings: Similarity significance value cutoff: >= 90%Summary of Percent Matches:Reference: human Smad7 mRNA 1-1281 (1281 bps) --Sequence 2: Tat-Smad7-V5 1-1392 (1392 bps) 68%

In this optimization, Met216, which may form an alternative open readingframe, was not altered as it was desired to preserve the amino acidsequence of Smad7, if possible. In future codon optimizations, Met216will be mutated to Leu216 to improve protein production withoutimpacting function in vitro and in vivo.

Example 7 Production of Truncated Smad7 Proteins

It is believed that Smad7 has several activities in vivo including, butnot limited to, one or more of enhancing cell proliferation, enhancingcell migration, reducing DNA damage, reducing cell apoptosis, anddecreasing inflammation. Smad7's effects on these processes are due toone or more of blocking TGF-3 signaling, blocking NF-κB signaling,blocking CtBP1 activity, and/or increasing Rac1 expression and/oractivity. It is believed that a smaller functional domain of Ptd-Smad7may be sufficient to deliver a therapeutic effect (see, e.g., FIG. 15).In addition, the resulting shorter protein sequence is expected toenhance protein production. Additionally, it is believed that differenttruncated Tat-Smad7 proteins that contain partial Smad7 sequences may beuseful for different treatments.

For example, it is believed that the C-terminal MH2 domain of Smad7(about half length of Smad7 protein, e.g., 208-426aa) may primarilymediate the anti-inflammatory effect of Smad7 (Hong et al., NatImmunology, 8, 504-513, 2007). Smad7 peptides having thisanti-inflammatory function may be sufficient and optionally animprovement for treating chronic inflammation associated conditions,such as but not limited to, oral mucositis, stomatitis and psoriasis,among others.

The N-terminal MH1 domain plus the linker region of Smad7 (about half ofthe protein, e.g., 2-208aa) is known to activate MAPK and binds toSmurf, a ubiquitin E3 ligase to degrade TGF-β receptor (Aragon, et al.,Structure 20:1726-1736 (2012)). It is believed that it may primarilymediate cell migration and/or blocking TGF-β-induced growth arrestand/or fibrotic response. Smad7 peptides having this cell migration andproliferation function may be sufficient, and optionally an improvement,for enhancing healing that is not associated with excessiveinflammation. Types of wounds that might benefit from this form oftreatment include, but are not limited to, surgical wounds, fibroticscarring, and diabetes wounds, defective healing and/or scarring amongothers.

Truncated Smad7 N-terminal and C-terminal PTD-fusion proteins weredesigned. One example of a Tat-Smad7-C-terminal codon-optimizednucleotide and protein sequence is provided below. In the nucleic acidsequence, nucleotides 1-6 include the restriction recognition site forBamHI; nucleotides 7-36 include the Tat PTD sequence; nucleotides 37-810include codon-optimized for the C terminal amino acids 258 to 426 ofhuman Smad7; nucleotides 568-609 include the V5 epitope sequence;nucleotides 610-612 include the stop sequence; and nucleotides 613-618include the restriction recognition site for SalI:

(SEQ ID NO: 24) ggatccggccgtaaaaaacgccgtcaacgccgccgttcacattggtgtgtggttgcctat (SEQ ID NO: 25) G S G R K K R R Q R R R S H W C V V A Ytgggaagagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgtc gctggatatcW E E K T R V G R L Y C V Q E P S L D Ittttatgacctgccgcagggcaatggtttctgtctgggccaactgaactc agataataaaF Y D L P Q G N G F C L G Q L N S D N Ktcgcagctggtgcaaaaagttcgctcaaaaattggctgcggtatccagct gacccgtgaaS Q L V Q K V R S K I G C G I Q L T R Egttgacggtgtctgggtatataaccgcagctcttacccgatttttatcaa aagtgccaccV D G V W V Y N R S S Y P I F I K S A Tctggataatccggactcccgtacgctgctggtccacaaagtatttccggg cttctcaatcL D N P D S R T L L V H K V F P G F S Iaaagcgttcgattacgagaaagcctactcgctgcagcgcccgaacgacca tgaattcatgK A F D Y E K A Y S L Q R P N D H E F Mcagcaaccgtggacgggttttactgtgcagatctctttcgttaaaggctg gggtcaatgcQ Q P W T G F T V Q I S F V K G W G Q Ctacacccgtcagtttatctcgtcctgtccgtgctggctggaagtgatttt caatagccgcY T R Q F I S S C P C W L E V I F N S Raagggcgagctcaattcgaagcttgaaggtaagcctatccctaaccctct cctcggtctcK G E L N S K L E G K P I P N P L L G L gattctacgtgagtcgac D S T

In the nucleic acid sequence, nucleotides 1-6 include the restrictionrecognition site for BamHI; nucleotides 7-36 include the Tat PTDsequence; nucleotides 37-810 include codon-optimized for the N terminalamino acids 1-258 of human Smad7; nucleotides 811-852 include the V5epitope sequence (corresponding amino acid sequence in bold);nucleotides 853-855 include the stop sequence; and nucleotides 856-861include the restriction recognition site for SalI. ATG is removed toallow for fusion with GST:

(SEQ ID NO: 26) ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgctcggcc (SEQ ID NO: 27) G S G R K K R R Q R R R G F R T K R S Actggtccgtcgcctgtggcgctcccgtgctccgggtggtgaagatgaaga agaaggtgctL V R R L W R S R A P G G E D E E E G Aggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatag tcgtgcacacG G G G G G G E L R G E G A T D S R A Hggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagc tgtgcgcggcG A G G G G P G R A G C C L G K A V R Ggcgaaaggtcatcaccatccgcacccgccggcagcaggtgcaggtgcagc tggcggtgcgA K G H H H P H P P A A G A G A A G G Agaagccgatctgaaagccctgacccatagtgtcctgaaaaaactgaaaga acgtcagctgE A D L K A L T H S V L K K L K E R Q Lgagctgctgctgcaagcagtagaatcccgtggcggtacccgtacggcttg tctgctgctgE L L L Q A V E S R G G T R T A C L L Lccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgca gccggcacaaP G R L D C R L G P G A P A G A Q P A Qccgccgagctcttacagcctgccgctgctgctgtgtaaagtgtttcgttg gccggacctgP P S S Y S L P L L L C K V F R W P D Lcgccacagttccgaagttaaacgcctgtgctgttgcgagagctatggcaa aattaacccgR H S S E V K R L C C C E S Y G K I N Pgaactggtttgttgcaatccgcaccatctgtctcgtctgtgtgaactgga gagcccgccgE L V C C N P H H L S R L C E L E S P Pccgccgtattctcgttacccgatggatttcctgaaaccgactgcagattg cccggacgcaP P Y S R Y P M D F L K P T A D C P D Agtcccgtcatcggctgagaccggcggcaccaactatctggcaccgggcgg tctgagtgatV P S S A E T G G T N Y L A P G G L S Dtcccagctgctgctggaaccgggcgaccgtggtaagcctatccctaaccc tctcctcggtS Q L L L E P G D R G K P I P N P L L G ctcgattctacgtgagtcgac L D S T

Example 8 Testing of Truncated Smad7 Proteins

Activity of truncated Smad7 proteins is tested using the in vitro and invivo assays used to test full-length Smad7 described above, among otherassays. Such assays include, but are not limited to, the ability toblock phosphorylation of Smad2 and/or nuclear translocation of the NF-κBp50 subunit, increase cell proliferation, reduce apoptosis and/orradiation-induced DNA damage, reduce inflammation and/or angiogenesis,promote healing in oral mucositis, surgical wounds, diabetes wounds,and/or wounds associated with chronic inflammation in mice.

In a wound healing assay, 6-mm punch biopsies were performed inwild-type mice followed by daily topical application of C-terminal orN-terminal Tat-Smad7. By measuring gross wound closure, both truncatedSmad7 proteins described above (e.g., Tat-Smad7 C-terminal andN-terminal protein) were found to have promoted wound healing similar tofull length Tat-Smad7.

Example 9 Truncated Smad7 Proteins Accelerate Wound Healing

The ability of truncated Smad7 proteins to accelerate wound healing wasexamined in wild-type mice. FIG. 16A demonstrates the effect ofC-terminally truncated (259-426aa) Tat-C-Smad7 on a mouse wound healingmodel. Wild-type C57BL/6 mice were anaesthetized, dorsally wounded by a6-mm dermal punch biopsy, and treated every other day with the topicalapplication of PBS (control, 3 mice, 4 wounds/mouse), full-lengthTat-Smad7 (0.4 μg/10 μL PBS/wound, 3 mice, 4 wounds/mouse) orTat-C-Smad7 (0.4 μg/10 μL/wound PBS, 3 mice, 4 wounds/mouse, total of 12wounds were treated). Wound area was photographed with Canon digitalcamera and measured by imaging analysis using the Image J software witha 6-mm circle inside of the photo for normalization at 1, 2, 4 and 5days after wounding, and the average percent wound area remaining wascalculated for each treatment group. The ability of Tat-C-Smad7 toaccelerate wound healing was similar to that of full-length Smad7 (FIG.16A).

FIG. 16B illustrates the effect of N-terminally truncated (1-258aa)Tat-N-Smad7 on wound healing. Wild-type C57BL/6 mice were anaesthetized,dorsally wounded by a 6-mm dermal punch biopsy, and treated every otherday with the topical application of PBS (control, 6 mice, 4wounds/mouse), full-length Tat-Smad7 (0.4 μg/10 μL PBS, 6 mice, 4wounds/mouse), or Tat-N-Smad7 (0.4 μg/10 μL PBS, 6 mice, 4wounds/mouse). Wound area was measured by imaging analysis using theImage J software with a 6-mm circle inside of the photo fornormalization at 1, 2, and 4 days after wounding, and the averagepercent wound area remaining was calculated for each treatment group.There was a significant difference in the rate of wound healing betweenthe control and Tat-Smad7-treated mice after two days, with the latterhealing faster (FIG. 16B, p<0.05). Although Tat-N-Smad7 was tested as anegative control, it unexpectedly promoted significantly acceleratedwound healing relative to control mice after just one day, andsignificantly accelerated wound healing relative to full-lengthTat-Smad7-treated mice after two days (FIG. 16B, p<0.05). Tat-Cre had noeffect on wound closure relative to control treatment (data not shown).

These results demonstrate that both full-length Tat-Smad7 and truncatedTat-Smad7 proteins (Tat-C-Smad7 and Tat-N-Smad7) promote wound healing.Moreover, these results demonstrate that certain truncated Tat-Smad7proteins are more effective in accelerating wound healing thanfull-length Tat-Smad7.

Accordingly, compositions comprising truncated Tat-Smad7 proteins areuseful in treating wounds and in accelerating wound healing.

Example 10 Smad7 Accelerates Wound Healing in an Impaired Wound-HealingModel

Diabetic (db/db) mice were dorsally wounded by a 6-mm dermal punchbiopsy as described above in Example 9, and treated every other day withthe topical application of PBS (control, 6 mice, 4 wounds/mouse),full-length Tat-Smad7 (0.4 μg/10 μL PBS, 6 mice, 4 wounds/mouse), orREGRANEX® cream (smear with cotton swab, 6 mice 4 wounds/mouse) to thewounds before day 8 when the wounds were not completely covered byscabs. After day 10, the treatments were topically applied to the gapbetween the scab and wound periphery to avoid the barrier of the hardscab and the unwounded stratum corneum. REGRANEX®, which contains arecombinant human platelet-derived growth factor (PDGF), is approved fortopical administration to diabetic ulcers.

Wound closure was visually assessed at 1, 2, 4, 6, 8, 9, 10, 11, 12, and13 days after wounding (FIG. 17A). At each time point, the wounds werephotographed with Canon digital camera. After 8 days, there was a markedvisual improvement in the wound closure in Tat-Smad7-treated micecompared to controls (FIG. 17A).

Wound size was measured by using the Image J software with a 6-mm circleinside of the photo for normalization at 1, 2, 3, 4, 7, 9, and 11 daysafter wounding, and the average percent wound area remaining wascalculated for each treatment group. The ability of Tat-Smad7 toaccelerate wound healing was similar to that of REGRANEX® (FIG. 17B). Onday 7, image analysis results indicated that wound closure wassignificantly accelerated in Tat-Smad7-treated mice relative to controls(FIG. 17B, p<0.05). These results were similar to those achieved withREGRANEX®.

Formalin fixed paraffin dorsal wound sections (1 mm) from day 8 woundsamples were stained using hematoxylin and eosin (H&E). Histologicalcomparison of day 8 wound samples revealed complete re-epithelializationand an accelerated wound closure in Smad7-treated db/db mice relative tocontrols (FIG. 17C).

Collectively, these results demonstrate that Tat-Smad7 is useful foraccelerating wound closure in poorly healing diabetic wounds, andprovides an alternative to treatment with REGRANEX®.

Example 11 Additional Codon Optimization for Smad7 Protein Production

Smad7 nucleic acid molecules are designed with additional nucleotidechanges selected to increase protein production. For example, theutilization of codons encoding the amino acids Ser and His will bemanipulated. In codon-optimized human Smad7 in examples above, the Sercodon (TCC or TCG) has an amino acid frequency of approximately 9% ofcodon utilization. It is believed that changing the codon of Ser to AGCwill increase Smad7 protein production, at least partly because it canoptionally increase codon usage to 15%. There are 33 Ser amino acids inSmad7 protein (nucleotides at positions 19-21, 46-48, 133-135, 292-294,349-351, 451-453, 454-456, 460-462, 511-513, 514-516, 544-546, 595-597,616-618, 634-636, 691-693, 694-696, 739-741, 745-747, 775-777, 847-849,907-909, 919-921, 943-945, 1006-1008, 1009-1101, 1030-1032, 1054-1056,1093-1095, 1126-1128, 1192-1194, 1237-1239, 1240-1242, 1273-1275; with acorresponding serine amino acid position of 7, 16, 45, 98, 117, 151,152, 154, 171, 172, 182, 199, 206, 212, 231, 232, 247, 249, 259, 283,303, 307, 315, 336, 337, 344, 352, 365, 376, 398, 413, 414, 425). Ofthese, 23 (nucleotides 19-21, 292-294, 349-351, 451-453, 454-456,460-462, 511-513, 514-516, 544-546, 616-618, 634-636, 691-693, 694-696,739-741, 745-747, 775-777, 847-849, 907-909, 919-921, 1009-1101,1030-1032, 1054-1056, 1093-1095; corresponding serine amino acidposition of 7, 98, 117, 151, 152, 154, 171, 172, 182, 206, 212, 231,232, 247, 249, 259, 283, 303, 307, 337, 344, 352, 365) can be changedwithout introducing potential alternative open reading frames.

Similarly, in codon-optimized human Smad7 in the examples above, the Hiscodon (CAC) has 9.6% of codon usage. It is believed that changing theHis codon to CAT (optionally to 12.6% usage) will increase Smad7 proteinproduction. There are 12 His (nucleotides 142-144, 214-216, 217-219,220-222, 226-228, 289-291, 589-591, 778-780, 1072-1074, 1147-1149;corresponding to histidine amino acids at position 48, 72, 73, 74, 76,97, 170, 196, 197, 260, 358, 383) in Smad7 protein. Of these, 4(nucleotides 217-219. 220-222, 589-591, 778-780, histidine residues 73,76, 197, 260) can be changed without introducing potential alternativeopen reading frames.

In addition, wild-type human Smad7 includes a Met amino acid as aminoacid 216 (Met216). This may be perceived as an alternative open readingframe by bacterial machinery, for example, and decrease proteinproduction. It is believed that changing Met216 to Leu216 (ATG to CTG),the amino acid that has the biochemical property the closest to Met andthus not expected to change 3D structure of the protein, will increaseprotein production.

The comparison between original codon-optimized Tat-Smad7-V5 and furtherchanges is provided below. Top strand: Tat-Smad7-V5 (SEQ ID NO: 23);bottom strand:

Alignment: Local DNA homologies.Parameters: Both strands. Method: FastScan - Max ScoreMol 1 20120323113102B12-S54366-164160-1-PGEX-5.seq (1-1463) Mol 2 TatSmad7 Ser-Hisoptim Number of sequences to align: 2Settings: Similarity significance value cutoff: >= 60%Homology Block: Percent Matches 94 Score 1227 Length 1392

201203231131  210 ggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggcTatSmad7 Ser  181 ............................................................

201203231131  450 ccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaaTatSmad7 Ser  421 ............................................................

201203231131 1410 acgtgagatcgac TatSmad7 Ser 1381 .............after optimized Ser, His and M216L mutation (SEQ ID NO: 30).

Nucleic acid sequences and their corresponding amino acid sequences thatwould include all these changes are provided below. The amino acidsequence includes the V5 epitope indicated in bold, and the pET101-Topobackbone indicated by italics and underlining. The Tat-Smad7^(M216L)fully-optimized full length nucleotide and protein sequence is shownbelow:

(SEQ ID NO: 30) ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgcagcgcc (SEQ ID NO: 31) G S G R K K R R Q R R R G F R T K R S Actggtccgtcgcctgtggcgcagccgtgctccgggtggtgaagatgaaga agaaggtgctL V R R L W R S R A P G G E D E E E G Aggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatag ccgtgcacatG G G G G G G E L R G E G A T D S R A Hggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagc tgtgcgcggcG A G G G G P G R A G C C L G K A V R Ggcgaaaggtcatcatcatccgcatccgccggcagcaggtgcaggtgcagc tggcggtgcgA K G H H H P H P P A A G A G A A G G Agaagccgatctgaaagccctgacccatagcgtcctgaaaaaactgaaaga acgtcagctgE A D L K A L T H S V L K K L K E R Q Lgagctgctgctgcaagcagtagaaagccgtggcggtacccgtacggcttg tctgctgctgE L L L Q A V E S R G G T R T A C L L Lccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgca gccggcacaaP G R L D C R L G P G A P A G A Q P A Qccgccgagcagctacagcctgccgctgctgctgtgtaaagtgtttcgttg gccggacctgP P S S Y S L P L L L C K V F R W P D Lcgccatagcagcgaagttaaacgcctgtgctgttgcgagagctatggcaa aattaacccgR H S S E V K R L C C C E S Y G K I N Pgaactggtttgttgcaatccgcatcatctgagccgtctgtgtgaactgga gagcccgccgE L V C C N P H H L S R L C E L E S P Pccgccgtatagccgttacccgctggatttcctgaaaccgactgcagattg cccggacgcaP P Y S R Y P L D F L K P T A D C P D Agtcccgagcagcgctgagaccggcggcaccaactatctggcaccgggcgg tctgagcgatV P S S A E T G G T N Y L A P G G L S Dagccagctgctgctggaaccgggcgaccgtagccattggtgtgtggttgc ctattgggaaS Q L L L E P G D R S H W C V V A Y W Egagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgagcctgga tatcttttatE K T R V G R L Y C V Q E P S L D I F Ygacctgccgcagggcaatggtttctgtctgggccaactgaacagcgataa taaaagccagD L P Q G N G F C L G Q L N S D N K S Qctggtgcaaaaagttcgcagcaaaattggctgcggtatccagctgacccg tgaagttgacL V Q K V R S K I G C G I Q L T R E V Dggtgtctgggtatataaccgcagcagctacccgatttttatcaaaagcgc caccctggatG V W V Y N R S S Y P I F I K S A T L Daatccggacagccgtacgctgctggtccataaagtatttccgggcttcag catcaaagcgN P D S R T L L V H K V F P G F S I K Attcgattacgagaaagcctacagcctgcagcgcccgaacgaccatgaatt catgcagcaaF D Y E K A Y S L Q R P N D H E F M Q Qccgtggacgggttttactgtgcagatcagcttcgttaaaggctggggtca atgctacaccP W T G F T V Q I S F V K G W G Q C Y Tcgtcagtttatcagcagctgtccgtgctggctggaagtgattttcaatag ccgcaagggcR Q F I S S C P C W L E V I F N S R  K Ggagctcaatagcaagcttgaaggtaagcctatccctaaccctctcctcgg tctcgatagcE L N S K L E  G K P I P N P L L G L D S acgtgagtcgac T

The optimized nucleotide and amino acid sequences will also be used tomake a variety of N-terminal and C-terminal Tat-Smad7 fragments.Representative examples are provided below.

The Tat-N-Smad7-V5 most optimized nucleotide and amino acid sequencesare provided. The protein sequence includes the V5 epitope, which isindicated by bold capital letters.

(SEQ ID NO: 32) ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgcagcgcc (SEQ ID NO: 27) G S G R K K R R Q R R R G F R T K R S Actggtccgtcgcctgtggcgcagccgtgctccgggtggtgaagatgaaga agaaggtgctL V R R L W R S R A P G G E D E E E G Aggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatag ccgtgcacatG G G G G G G E L R G E G A T D S R A Hggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagc tgtgcgcggcG A G G G G P G R A G C C L G K A V R Ggcgaaaggtcatcatcatccgcatccgccggcagcaggtgcaggtgcagc tggcggtgcgA K G H H H P H P P A A G A G A A G G Agaagccgatctgaaagccctgacccatagcgtcctgaaaaaactgaaaga acgtcagctgE A D L K A L T H S V L K K L K E R Q Lgagctgctgctgcaagcagtagaaagccgtggcggtacccgtacggcttg tctgctgctgE L L L Q A V E S R G G T R T A C L L Lccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgca gccggcacaaP G R L D C R L G P G A P A G A Q P A Qccgccgagcagctacagcctgccgctgctgctgtgtaaagtgtttcgttg gccggacctgP P S S Y S L P L L L C K V F R W P D Lcgccatagcagcgaagttaaacgcctgtgctgttgcgagagctatggcaa aattaacccgR H S S E V K R L C C C E S Y G K I N Pgaactggtttgttgcaatccgcatcatctgagccgtctgtgtgaactgga gagcccgccgE L V C C N P H H L S R L C E L E S P Pccgccgtatagccgttacccgatggatttcctgaaaccgactgcagattg cccggacgcaP P Y S R Y P M D F L K P T A D C P D Agtcccgagcagcgctgagaccggcggcaccaactatctggcaccgggcgg tctgagcgatV P S S A E T G G T N Y L A P G G L S Dagccagctgctgctggaaccgggcgaccgtggtaagcctatccctaaccc tctcctcggtS Q L L L E P G D R G K P I P N P L L G ctcgattctacgtgagtcgac L D S T

Tat-C-Smad7-V5 most optimized nucleotide and amino acid sequences areprovided. The protein sequence includes the V5 epitope (indicated bybold capital letters), and the pET101-Topo backbone (indicated byunderlined italics).

(SEQ ID NO: 34) ggatccggccgtaaaaaacgccgtcaacgccgccgttcacattggtgtgtggttgcctat (SEQ ID NO: 25) G S G R K K R R Q R R R S H W C V V A Ytgggaagagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgag cctggatatcW E E K T R V G R L Y C V Q E P S L D Ittttatgacctgccgcagggcaatggtttctgtctgggccaactgaacag cgataataaaF Y D L P Q G N G F C L G Q L N S D N Kagccagctggtgcaaaaagttcgcagcaaaattggctgcggtatccagct gacccgtgaaS Q L V Q K V R S K I G C G I Q L T R Egttgacggtgtctgggtatataaccgcagcagctacccgatttttatcaa aagcgccaccV D G V W V Y N R S S Y P I F I K S A Tctggataatccggacagccgtacgctgctggtccataaagtatttccggg cttcagcatcL D N P D S R T L L V H K V F P G F S Iaaagcgttcgattacgagaaagcctacagcctgcagcgcccgaacgacca tgaattcatgK A F D Y E K A Y S L Q R P N D H E F Mcagcaaccgtggacgggttttactgtgcagatcagcttcgttaaaggctg gggtcaatgcQ Q P W T G F T V Q I S F V K G W G Q Ctacacccgtcagtttatcagcagctgtccgtgctggctggaagtgatttt caatagccgcY T R Q F I S S C P C W L E V I F N S Raagggcgagctcaatagcaagettgaaggtaagcctatccctaaccctct cctcggtctcK G E L N S K L E  G K P I P N P L L G L gatagcacgtgagtcgac D S T

The comparisons before and after the above optimizations are providedbelow. C-terminal optimization (top strand) (alignment discloses SEQ IDNOs: 34 and 24, respectively, in order of appearance):

Alignment: Local DNA homologies.Parameters: Both strands. Method: FastScan - Max ScoreMol 1 Tat-C-Smad7-ser-his optimized (1-618) Mol 2 Tat-C-termal Smad7-V5 (1-618)Number of sequences to align: 2Settings: Similarity significance value cutoff: >= 60%Homology Block: Percent Matches 93 Score 541 Length 618Tat-C-Smad7-   1 ggatccggccgtaaaaaacgccgtcaacgccgccgttcacattggtgtgtggttgcctatTat-C-termal   1 ............................................................

N-terminal optimization (top strand) (alignment discloses SEQ ID NOs: 32and 26, respectively, in order of appearance):

Alignment: Local DNA homologies.Parameters: Both strands. Method: FastScan - Max ScoreMol 1 Tat-N-Smad7-V5-Ser-His optimized (1-861) Mol 2 Tat-N-Smad7-V5 (1-861)Number of sequences to align: 2Settings: Similarity significance value cutoff: >= 60%Homology Block: Percent Matches 95 Score 781 Length 861

Tat-N-Smad7- 181 ggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggcTat-N-Smad7- 181 ............................................................

Tat-N-Smad7- 421 ccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaaTat-N-Smad7- 421 ............................................................

Tat-N-Smad7- 841 ctcgattctacgtgagtcgacTat-N-Smad7- 841 .....................

In addition, other codon-optimized nucleic acids will also be assessedfor their ability to produce Smad7 protein in one or more expressionsystems. Provided below is another example of such a sequence.

Tat-Smad7^(M7216L)-V5 optimized by Optimizer program:

(SEQ ID NO: 36) ggatccggtcgtaaaaaacgtcgtcagcgtcgtcgtggtttccgtaccaaacgttctgcg (SEQ ID NO: 37) G S G R K K R R Q R R R G F R T K R S Actggttcgtcgtctgtggcgttctcgtgcgccgggtggtgaagacgaaga agaaggtgcgL V R R L W R S R A P G G E D E E E G Aggtggtggtggtggtggtggtgaactgcgtggtgaaggtgcgaccgactc tcgtgcgcacG G G G G G G E L R G E G A T D S R A Hggtgcgggtggtggtggtccgggtcgtgcgggttgctgcctgggtaaagc ggttcgtggtG A G G G G P G R A G C C L G K A V R Ggcgaaaggtcaccaccacccgcacccgccggcggcgggtgcgggtgcggc gggtggtgcgA K G H H H P H P P A A G A G A A G G Agaagcggacctgaaagcgctgacccactctgttctgaaaaaactgaaaga acgtcagctgE A D L K A L T H S V L K K L K E R Q Lgaactgctgctgcaggcggttgaatctcgtggtggtacccgtaccgcgtg cctgctgctgE L L L Q S R G G T R T A C L L L L L Lccgggtcgtctggactgccgtctgggtccgggtgcgccggcgggtgcgca gccggcgcagP G R L D C R L G P G A P A G A Q P A Qccgccgtcttcttactctctgccgctgctgctgtgcaaagttttccgtt ggccggacctgP P S S Y S L P L L L C K V F R W P D Lcgtcactcttctgaagttaaacgtctgtgctgctgcgaatcttacggta aaatcaacccgR H S S E V K R L C C C E S Y G K I N Pgaactggtttgctgcaacccgcaccacctgtctcgtctgtgcgaactgg aatctccgccgE L V C C N P H H L S R L C E L E S P Pccgccgtactctcgttacccgctggacttcctgaaaccgaccgcggact gcccggacgcgP P Y S R Y P L D F L K P T A D C P D Agttccgtcttctgcggaaaccggtggtaccaactacctggcgccgggtg gtctgtctgacV P S S A E T G G T N Y L A P G G L S Dtctcagctgctgctggaaccgggtgaccgttctcactggtgcgttgttg cgtactgggaaS Q L L L E P G D R S H W C V V A Y W Egaaaaaacccgtgttggtcgtctgtactgcgttcaggaaccgtctctgg acatcttctacE K T R V G R L Y C V Q E P S L D I F Ygacctgccgcagggtaacggtttctgcctgggtcagctgaactctgaca acaaatctcagD L P Q G N G F C L G Q L N S D N K S Qctggttcagaaagttcgttctaaaatcggttgcggtatccagctgaccc gtgaagttgacL V Q K V R S K I G C G I Q L T R E V Dggtgtttgggtttacaaccgttcttcttacccgatcttcatcaaatctg cgaccctggacG V W V Y N R S S Y P I F I K S A T L Daacccggactctcgtaccctgctggttcacaaagttttcccgggtttct ctatcaaagcgN P D S R T L L V H K V F P G F S I K Attcgactacgaaaaagcgtactctctgcagcgtccgaacgaccacgaat tcatgcagcagF D Y E K A Y S L Q R P N D H E F M Q Qccgtggaccggtttcaccgttcagatctctttcgttaaaggttggggtc agtgctacaccP W T G F T V Q I S F V K G W G Q C Y Tcgtcagttcatctcttcttgcccgtgctggctggaagttatcttcaact ctcgtggtaaaR Q F I S S C P C W L E V I F N S R G Kccgatcccgaacccgctgctgggtctggactctacctgagtcgac P I P N P L L G L D S T

Nucleotide sequence:

1-6: BamHI; 7-36: Tat; 37-1314: codon-optimized human Smad7; 1315-1356: V5;137-1359: stop; 1360-1365 SalIATG is removed to be used with GST; 682 ATG to CTG (M216 to L)(SEQ ID NO: 36)    1ggatccggtc gtaaaaaacg tcgtcagcgt cgtcgtggtt tccgtaccaa acgttctgcg   61ctggttcgtc gtctgtggcg ttctcgtgcg ccgggtggtg aagacgaaga agaaggtgcg  121ggtggtggtg gtggtggtgg tgaactgcgt ggtgaaggtg cgaccgactc tcgtgcgcac  181ggtgcgggtg gtggtggtcc gggtcgtgcg ggttgctgcc tgggtaaagc ggttcgtggt  241gcgaaaggtc accaccaccc gcacccgccg gcggcgggtg cgggtgcggc gggtggtgcg  301gaagcggacc tgaaagcgct gacccactct gttctgaaaa aactgaaaga acgtcagctg  361gaactgctgc tgcaggcggt tgaatctcgt ggtggtaccc gtaccgcgtg cctgctgctg  421ccgggtcgtc tggactgccg tctgggtccg ggtgcgccgg cgggtgcgca gccggcgcag  481ccgccgtctt cttactctct gccgctgctg ctgtgcaaag ttttccgttg gccggacctg  541cgtcactctt ctgaagttaa acgtctgtgc tgctgcgaat cttacggtaa aatcaacccg  601gaactggttt gctgcaaccc gcaccacctg tctcgtctgt gcgaactgga atctccgccg  661ccgccgtact ctcgttaccc gctggacttc ctgaaaccga ccgcggactg cccggacgcg  721gttccgtctt ctgcggaaac cggtggtacc aactacctgg cgccgggtgg tctgtctgac  781tctcagctgc tgctggaacc gggtgaccgt tctcactggt gcgttgttgc gtactgggaa  841gaaaaaaccc gtgttggtcg tctgtactgc gttcaggaac cgtctctgga catcttctac  901gacctgccgc agggtaacgg tttctgcctg ggtcagctga actctgacaa caaatctcag  961ctggttcaga aagttcgttc taaaatcggt tgcggtatcc agctgacccg tgaagttgac 1021ggtgtttggg tttacaaccg ttcttcttac ccgatcttca tcaaatctgc gaccctggac 1081aacccggact ctcgtaccct gctggttcac aaagttttcc cgggtttctc tatcaaagcg 1141ttcgactacg aaaaagcgta ctctctgcag cgtccgaacg accacgaatt catgcagcag 1201ccgtggaccg gtttcaccgt tcagatctct ttcgttaaag gttggggtca gtgctacacc 1261cgtcagttca tctcttcttg cccgtgctgg ctggaagtta tcttcaactc tcgtggtaaa 1321ccgatcccga acccgctgct gggtctggac tctacctgag tcgac

Sequence comparison with Tat-Smad7^(M7216L)-V5 described in Example 4(alignment discloses SEQ ID NOs: 36 and 30, respectively, in order ofappearance):

Alignment: Global DNA alignment against reference moleculeParameters: Scoring matrix: Linear (Mismatch 2, OpenGap 4, ExtGap 1)Reference molecule: Tat-Smad7-216L-V5-optimizer, Region 1-1365Number of sequences to align: 2Settings: Similarity significance value cutoff: >= 60%Summary of Percent Matches:Reference: Tat-Smad7-216L-V5-optimizer 1-1365 (1365 bps) --Sequence 2: TatSmad7 Ser-His optimized-682 mutant 1-1392 (1392 bps) 79%

Protein production using the sequences below is carried out bytransfecting mammalian expression vectors (e.g., pCMV-6-Entry) intoHEK293 or CHO cells.

Optimized GST-Tat-Smad7-myc-flag nucleotide sequence, cloned inpCMV6-Entry plasmid:

(SEQ ID NO: 87)    1gacgttgtat acgactccta tagggcggcc gggaattcgt cgactggatc cggtaccgag   61gagatctgcc gccgcgatcg ccatgtcccc cattctgggc tactggaaga ttaagggcct  121ggtgcagcct actagactgc tgctggaata cctggaggaa aaatatgaag agcatctgta  181tgaaagagac gagggggata aatggaggaa caagaaattc gaactgggac tggagtttcc  241taatctgcca tactatattg acggcgatgt gaagctgact cagtctatgg ctatcattag  301atacatcgca gacaaacaca acatgctggg cgggtgtcct aaggaaaggg cagagattag  361tatgctggag ggagccgtgc tggatattag atacggcgtc tcacgcatcg cctatagcaa  421agacttcgaa accctgaagg tggattttct gagcaaactg cctgaaatgc tgaagatgtt  481cgaggacaga ctgtgccaca aaacctacct gaatggcgac catgtcacac acccagattt  541tatgctgtac gacgccctgg atgtggtcct gtatatggac cccatgtgtc tggatgcttt  601ccctaagctg gtgtgcttta agaaaaggat cgaggcaatt ccccagatcg ataagtacct  661gaaaagctcc aagtatatcg cttggcctct ccagggctgg caggcaacat tcggaggcgg  721ggaccatccc cctaaaagcg acctggaggt gctgtttcag ggaccactgg gcagcggccg  781gaagaagcgg cggcagaggc gccgaagtag gttccgcact aagcggtcag cactggtgcg  841gagactgtgg cgatctcggg ctcctggagg agaggacgag gaagagggag caggcggcgg  901cggaggagga ggagaactgc gcggggaggg agctacagat agccgagccc acggagctgg  961aggaggagga ccagggcgag ccggatgctg tctgggcaaa gcagtgagag gcgccaaggg 1021gcaccatcac ccccatccac ccgccgctgg cgcaggagca gccggcggag ctgaggcaga 1081cctgaaagcc ctgactcaca gtgtgctgaa gaaactgaag gaaagacagc tggagctgct 1141gctccaggca gtcgaatcac gcggaggcac ccgaacagct tgtctgctgc tgcccggccg 1201gctggactgc cggctgggac ccggcgcccc tgctggggca cagccagccc agcctccatc 1261tagttatagc ctgcccctgc tgctgtgtaa ggtgttccga tggcctgatc tgcggcattc 1321aagcgaagtc aaaaggctgt gctgttgcga gtcctacggc aagattaacc cagaactggt 1381gtgttgcaat ccccatcacc tgtctcgact gtgtgaactg gagtcccccc ctccacccta 1441ctctaggtat cctatggact ttctgaagcc aaccgctgac tgcccagatg cagtgccctc 1501ctctgccgag actgggggaa ccaactacct ggctcctggc ggactgagcg actcccagct 1561gctgctggaa ccaggggatc gcagccactg gtgtgtggtc gcctactggg aagagaagac 1621aagagtggga aggctgtatt gcgtccagga gccttccctg gacatcttct acgatctgcc 1681acaggggaat ggattttgtc tgggccagct gaactctgac aataagagtc agctggtgca 1741gaaagtccgg agcaagattg gctgcggcat ccagctgacc agggaggtgg acggcgtgtg 1801ggtctacaac cgcagttcat atccaatctt catcaagagc gccactctgg acaatcccga 1861ttcccgcacc ctgctggtgc ataaggtctt ccccggcttc agcatcaagg ccttcgacta 1921cgagaaggct tatagtctcc agcggcccaa cgatcacgag ttcatgcagc agccttggac 1981aggcttcact gtgcagatca gcttcgtcaa gggatggggc cagtgctaca caaggcagtt 2041catctcaagc tgtccctgtt ggctggaagt cattttcaat agtaggacgc gtac g c gg cc2101 g c tcgagcag aaactcatct cagaagagga tctggcagca aatgatatcc tggattacaa2161 ggatgacgac gataaggttt aa

The entire sequence (up to the NotI site) is codon optimized formammalian expression, using the OPTIMUMGENE™ program from GenScript.

1790-1792: changes GAT to GAC, (kept Asp) to avoid alternative ORF(highlight)

1-82: cloning sites+kozac sequence in pCMV6-Entry

83-777: GST+precision enzyme sites; 776-805: Tat;

806-2086: full length human Smad7

2087-2107: MluI, NotI, XhoI

2108-2182: myc-flag from pCMV6-Entry

Bold underline: unique NotI site

The above nucleotide sequence is 79% homologous to native human Smad7.

Mol 1 human Smad7 mRNA (1-3103) Mol 2 optimized GST-Tat-Smad7-myc-flag(1-2182) Number of sequences to align: 2Settings: Sort results list by Score human Smad7  500tcaccaccatccccacccgccagccgcgggcgccggcgcggccgggggcg optimized GS 1027.....c......--.......----..t.....a..a..a.....c..a. human Smad7  550ccgaggcggatctgaaggcgctcacgcactcggtgctcaagaaactgaag optimized GS 1071.t.....a..c.....a..c..g..t...agt.....g............ human Smad7  600gagcggcagctggagctgctgctccaggccgtggagtcccgcggcgggac optimized GS 1121..aa.a.......................a..c..a..a.....a..c.. human Smad7  650gcgcaccgcgtgcctcctgctgcccggccgcctggactgcaggctgggcc optimized GS 1171c..a..a..t..t..g..............g.........c.......a. human Smad7  700cgggggcgcccgccggcgcgcagcctgcgcagccgccctcgtcctactcg optimized GS 1221.c..c..c..t..t..g..a.....a..c.....t..a..tagt..tagc human Smad7  750ctccccctcctgctgtgcaaagtgttcaggtggccggatctcaggcattc optimized GS 1271..g.....g........t..g......c.a.....t.....gc....... human Smad7  800ctcggaagtcaagaggctgtgttgctgtgaatcttacgggaagatcaacc optimized GS 1321aagc........a........c..t..c..g..c.....c.....t.... human Smad7  850ccgagctggtgtgctgcaacccccatcaccttagccgactctgcgaacta optimized GS 1371.a..a........t.....t...........gtct.....g..t.....g human Smad7  900gagtctcccccccctccttactccagatacccgatggattttctcaaacc optimized GS 1421.....c.....t..a..c.....t..g..t..t.....c.....g..g.. human Smad7  950aactgcagactgtccagatgctgtgccttcctccgctgaaacagggggaa optimized GS 1471...c..t.....c........a.....c.....t..c..g..t....... human Smad7 1000cgaattatctggcccctggggggctttcagattcccaacttcttctggag optimized GS 1521.c..c..c.....t.....c..a..gagc..c.....g..g..g.....a human Smad7 1050cctggggatcggtcacactggtgcgtggtggcatactgggaggagaagac optimized GS 1571..a........cagc........t.....c..c........a........ human Smad7 1100gagagtggggaggctctactgtgtccaggagccctctctggatatcttct optimized GS 1621a........a.....g..t..c...........t..c.....c....... human Smad7 1150atgatctacctcaggggaatggcttttgcctcggacagctcaattcggac optimized GS 1671.c.....g..a...........a.....t..g..c.....g..c..t... human Smad7 1200aacaagagtcagctggtgcagaaggtgcggagcaaaatcggctgcggcat optimized GS 1721..t....................a..c........g..t........... human Smad7 1250ccagctgacgcgggaggtggatggtgtgtgggtgtacaaccgcagcagtt optimized GS 1771.........ca..........c..c........c...........ttca. human Smad7 1300accccatcttcatcaagtccgccacactggacaacccggactccaggacg optimized GS 1821.t..a............ag......t........t..c..t...c.c..c human Smad7 1350ctgttggtacacaaggtgttccccggtttctccatcaaggctttcgacta optimized GS 1871...c....g..t.....c........c...ag.........c........ human Smad7 1400cgagaaggcgtacagcctgcagcggcccaatgaccacgagtttatgcagc optimized GS 1921.........t..t..t..c...........c..t........c....... human Smad7 1450agccgtggacgggctttaccgtgcagatcagctttgtgaagggctggggt optimized GS 1971....t.....a.....c..t..............c..c.....a.....c human Smad7 1500cagtgctacacccgccagttcatcagcagctgcccgtgctggctagaggt optimized GS 2021...........aa.g.........tca.....t..c..t.....g..a.. human Smad7 1550catcttcaa (SEQ ID NO: 88) optimized GS 2071 ...t..... (SEQ ID NO: 89)

Optimized GST-Tat-Smad7-myc-flag protein sequence from the abovenucleotide sequence:

(SEQ ID NO: 90)   1mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl efpnlpyyid  61gdvkltqsma iiryiadkhn mlggcpkera eismlegavl dirygvsria yskdfetlkv 121dflsklpeml kmfedrlchk tylngdhvth pdfmlydald vvlymdpmcl dafpklvcfk 181krieaipqid kylksskyia wplqgwqatf gggdhppksd levlfqgplg sgrkkrrqrr 241rsrfrtkrsa lvrrlwrsra pggedeeega gggggggelr gegatdsrah gaggggpgra 301gcclgkavrg akghhhphpp aagagaagga eadlkalths vlkklkerql elllqavesr 361ggtrtaclll pgrldcrlgp gapagaqpaq ppssyslpll lckvfrwpdl rhssevkrlc 421ccesygkinp elvccnphhl srlcelespp ppysrypmdf lkptadcpda vpssaetggt 481nylapgglsd sqlllepgdr shwcvvaywe ektrvgrlyc vqepsldify dlpqgngfcl 541gqlnsdnksq lvqkvrskig cgiqltrevd gvwvynrssy pifiksatld npdsrtllvh 601kvfpgfsika fdyekayslq rpndhefmqq pwtgftvqis fvkgwgqcyt rqfisscpcw 661levifnsrtr trpleqklis eedlaandil dykddddkv

1-229: GST+precision sites

232-668 Tat-Smad7

671-699: restriction sites+myc-Flag

Optimized GST-Tat-N-Smad7-myc-flag nucleotide sequence, cloned inpCMV6-Entry plasmid:

(SEQ ID NO: 91)    1gaattcgtcg actggatccg gtaccgagga gatctgccgc cgcgatcgcc atgtccccca   61ttctgggcta ctggaagatt aagggcctgg tgcagcctac tagactgctg ctggaatacc  121tggaggaaaa atatgaagag catctgtatg aaagagacga gggggataaa tggaggaaca  181agaaattcga actgggactg gagtttccta atctgccata ctatattgac ggcgatgtga  241agctgactca gtctatggct atcattagat acatcgcaga caaacacaac atgctgggcg  301ggtgtcctaa ggaaagggca gagattagta tgctggaggg agccgtgctg gatattagat  361acggcgtctc acgcatcgcc tatagcaaag acttcgaaac cctgaaggtg gattttctga  421gcaaactgcc tgaaatgctg aagatgttcg aggacagact gtgccacaaa acctacctga  481atggcgacca tgtcacacac ccagatttta tgctgtacga cgccctggat gtggtcctgt  541atatggaccc catgtgtctg gatgctttcc ctaagctggt gtgctttaag aaaaggatcg  601aggcaattcc ccagatcgat aagtacctga aaagctccaa gtatatcgct tggcctctcc  661agggctggca ggcaacattc ggaggcgggg accatccccc taaaagcgac ctggaggtgc  721tgtttcaggg accactgggc agcggccgga agaagcggcg gcagaggcgc cgaagtaggt  781tccgcactaa gcggtcagca ctggtgcgga gactgtggcg atctcgggct cctggaggag  841aggacgagga agagggagca ggcggcggcg gaggaggagg agaactgcgc ggggagggag  901ctacagatag ccgagcccac ggagctggag gaggaggacc agggcgagcc ggatgctgtc  961tgggcaaagc agtgagaggc gccaaggggc accatcaccc ccatccaccc gccgctggcg 1021caggagcagc cggcggagct gaggcagacc tgaaagccct gactcacagt gtgctgaaga 1081aactgaagga aagacagctg gagctgctgc tccaggcagt cgaatcacgc ggaggcaccc 1141gaacagcttg tctgctgctg cccggccggc tggactgccg gctgggaccc ggcgcccctg 1201ctggggcaca gccagcccag cctccatcta gttatagcct gcccctgctg ctgtgtaagg 1261tgttccgatg gcctgatctg cggcattcaa gcgaagtcaa aaggctgtgc tgttgcgagt 1321cctacggcaa gattaaccca gaactggtgt gttgcaatcc ccatcacctg tctcgactgt 1381gtgaactgga gtccccccct ccaccctact ctaggtatcc tatggacttt ctgaagccaa 1441ccgctgactg cccagatgca gtgccctcct ctgccgagac tgggggaacc aactacctgg 1501ctcctggcgg actgagcgac tcccagctgc tgctggaacc aggggatcg g   cggccgc tcg1561 agcagaaact catctcagaa gaggatctgg cagcaaatga tatcctggat tacaaggatg1621 acgacgataa ggtttaa

The entire sequence (up to the NotI site) is codon optimized formammalian expression, using the OPTIMUMGENE™ program from GenScript.

1-50: cloning sites+kozac sequence in pCMV6-Entry

51-743: GST+precision enzyme sites

744-773-: Tat

774-1549-: human Smad7 encoding N-terminal 1-258aa

1550-1563: NotI, XhoI, Bold underline: unique NotI site

1564-1637: myc-flag from pCMV6-Entry

Optimized GST-Tat-N-Smad7-myc-flag protein sequence from the abovenucleotide sequence:

(SEQ ID NO: 92)   1mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl efpnlpyyid  61gdvkltqsma iiryiadkhn mlggcpkera eismlegavl dirygvsria yskdfetlkv 121dflsklpeml kmfedrlchk tylngdhvth pdfmlydald vvlymdpmcl dafpklvcfk 181krieaipqid kylksskyia wplqgwqatf gggdhppksd levlfqgplg sgrkkrrqrr 241rsrfrtkrsa lvrrlwrsra pggedeeega gggggggelr gegatdsrah gaggggpgra 301gcclgkavrg akghhhphpp aagagaagga eadlkalths vlkklkerql elllqavesr 361ggtrtaclll pgrldcrlgp gapagaqpaq ppssyslpll lckvfrwpdl rhssevkrlc 421ccesygkinp elvccnphhl srlcelespp ppysrypmdf lkptadcpda vpssaetggt 481nylapgglsd sqlllepgdr rpleqklise edlaandild ykddddkv

1-241: GST-Tat

244-500: N-Smad7 2-258

501-528: restriction sites+myc-Flag

Optimized GST-Tat-C-Smad7-myc-flag nucleotide sequence, cloned inpCMV6-Entry plasmid:

(SEQ ID NO: 93)    1

  61 ttctgggcta ctggaagatt aagggcctgg tgcagcctac tagactgctg ctggaatacc 121 tggaggaaaa atatgaagag catctgtatg aaagagacga gggggataaa tggaggaaca 181 agaaattcga actgggactg gagtttccta atctgccata ctatattgac ggcgatgtga 241 agctgactca gtctatggct atcattagat acatcgcaga caaacacaac atgctgggcg 301 ggtgtcctaa ggaaagggca gagattagta tgctggaggg agccgtgctg gatattagat 361 acggcgtctc acgcatcgcc tatagcaaag acttcgaaac cctgaaggtg gattttctga 421 gcaaactgcc tgaaatgctg aagatgttcg aggacagact gtgccacaaa acctacctga 481 atggcgacca tgtcacacac ccagatttta tgctgtacga cgccctggat gtggtcctgt 541 atatggaccc catgtgtctg gatgctttcc ctaagctggt gtgctttaag aaaaggatcg 601 aggcaattcc ccagatcgat aagtacctga aaagctccaa gtatatcgct tggcctctcc 661 agggctggca ggcaacattc ggaggcgggg accatccccc taaaagcgac ctggaggtgc 721 tgtttcaggg accactgggc agcggccgga agaagcggcg gcagaggcgc agccactggt 781 gtgtggtcgc ctactgggaa gagaagacaa gagtgggaag gctgtattgc gtccaggagc 841 cttccctgga catcttctac gatctgccac aggggaatgg attttgtctg ggccagctga 901 actctgacaa taagagtcag ctggtgcaga aagtccggag caagattggc tgcggcatcc 961 agctgaccag ggaggtggac ggcgtgtggg tctacaaccg cagttcatat ccaatcttca1021 tcaagagcgc cactctggac aatcccgatt cccgcaccct gctggtgcat aaggtcttcc1081 ccggcttcag catcaaggcc ttcgactacg agaaggctta tagtctccag cggcccaacg1141 atcacgagtt catgcagcag ccttggacag gcttcactgt gcagatcagc ttcgtcaagg1201 gatggggcca gtgctacaca aggcagttca tctcaagctg tccctgttgg ctggaagtca1261

1321 tggcagcaaa tgatatcctg gattacaagg atgacgacga taaggtttaa

The entire sequence (up to the NotI site) is codon optimized formammalian expression, using the OPTIMUMGENE™ program from GenScript.

1-50: cloning sites+kozac sequence in pCMV6-Entry

51-743: GST+precision enzyme sites

744-773: Tat

774-1274: c-terminal Smad7

1275: Mlu

1283: NotI

1275-1370: mluI, NotI, XhoI, myc+flag

Optimized GST-Tat-C-Smad7-myc-flag protein sequence from the abovenucleotide sequence:

(SEQ ID NO: 94)   1mspilgywki kglvqptrll leyleekyee hlyerdegdk wrnkkfelgl efpnlpyyid  61gdvkltqsma iiryiadkhn mlggcpkera eismlegavl dirygvsria yskdfetlkv 121dflsklpeml kmfedrlchk tylngdhvth pdfmlydald vvlymdpmcl dafpklvcfk 181krieaipqid kylksskyia wplqgwqatf gggdhppksd levlfqgplg sgrkkrrqrr 241rshwcvvayw eektrvgrly cvqepsldif ydlpqgngfc lgqlnsdnks qlvqkvrski 301gcgiqltrev dgvwvynrss ypifiksatl dnpdsrtllv hkvfpgfsik afdyekaysl 361qrpndhefmq qpwtgftvqi sfvkgwgqcy trqfisscpc wlevifnsrt rtrpleqkli 421seedlaandi ldykddddkv

1-241: GST+precision+Tat

242-409: c-Smad7 (259-426aa)

410-440: Mlu+Not+myc+flag: restriction sites+myc-Flag

Based on the Smad7 structure, it is anticipated that the linker(203-258aa), which contains the PY domain (203-217aa), will havetherapeutic effects by blocking TGF-β-induced inflammation or growthinhibition (FIG. 15).

The amino acid sequence for the Tat-Smad7-linker peptide (203-258aa) is:

(SEQ ID NO: 95) GRKKRRQRRR-ELESPPPPYSRYPMDFLKPTADCPDAVPSSAETGGTNYLAPGGLSDSQLL LEPGDR

1-10: Tat

The codon optimized nucleotide sequence for the above peptide inbacterial production is:

(SEQ ID NO: 96)   1ggccgtaaaa aacgccgtca acgccgccgt gaactggaga gcccgccgcc gccgtatagc  61cgttacccga tggatttcct gaaaccgact gcagattgcc cggacgcagt cccgagcagc 121gctgagaccg gcggcaccaa ctatctggca ccgggcggtc tgagcgatag ccagctgctg 181ctggaaccgg gcgaccgtgg taagcctatc cctaaccctc tcctcggtct cgattctacg 241tgagtcgac

1-30: Tat

31-198: Smad7 linker

199-249: V5

The codon optimized nucleotide sequence for the above peptide inmammalian cell production is:

(SEQ ID NO: 97)   1ggccggaaga agcggcggca gaggcgccga gaactggagt ccccccctcc accctactct  61aggtatccta tggactttct gaagccaacc gctgactgcc cagatgcagt gccctcctct 121gccgagactg ggggaaccaa ctacctggct cctggcggac tgagcgactc ccagctgctg 181ctggaaccag gggatcggcg gccgctcgag cagaaactca tctcagaaga ggatctggca 241gcaaatgata tcctggatta caaggatgac gacgataagg tttaa

1-30: mammalian codon optimized Tat

31-198: Smad7 linker

199-285: NotI, Myc tag, Flag tag

The amino acid sequence for the Tat-Smad7-PY peptide (203-217aa) is:

GRKKRRQRRR-ELESPPPPYSRYPMD  (SEQ ID NO: 98)

1-10: Tat

The codon optimized nucleotide sequence for the above peptide inbacterial production is:

(SEQ ID NO: 99)   1ggccgtaaaa aacgccgtca acgccgccgt gaactggaga gcccgccgcc gccgtatagc  61cgttacccga tggatggtaa gcctatccct aaccctctcc tcggtctcga ttctacgtga 121gtcgac

1-30: Tat

31-75: Smad7-PY

76-126: V5

The codon optimized nucleotide sequence for the above peptide inmammalian cell production is:

(SEQ ID NO: 100)   1ggccggaaga agcggcggca gaggcgccga gaactggagt ccccccctcc accctactct  61aggtatccta tggaccggcc gctcgagcag aaactcatct cagaagagga tctggcagca 121aatgatatcc tggattacaa ggatgacgac gataaggttt aa

1-30: mammalian codon optimized Tat

31-75: Smad7 PY

76-162: NotI, Myc tag, Flag tag

The foregoing discussion of the present technology has been presentedfor purposes of illustration and description. The foregoing is notintended to limit the present technology to the form or forms disclosedherein. Although the description of the present technology has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the present technology, e.g., as may be within the skill andknowledge of those in the art, after understanding the presentdisclosure. It is intended to obtain rights which include alternativeembodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1.-12. (canceled)
 13. A nucleic acid molecule comprising a codon-optimized cDNA nucleotide sequence comprising nucleotides encoding amino acids 204-258 of the human Smad7 protein.
 14. The nucleic acid molecule of claim 13, wherein the nucleotide sequence further comprises a nucleotide sequence encoding a protein transduction domain.
 15. The nucleic acid molecule of claim 14, wherein the protein transduction domain is Tat. 16.-19. (canceled)
 20. The nucleic acid of claim 13, wherein the nucleic acid molecule encodes a polypeptide having one or more biological activity selected from the group consisting of promoting healing in oral mucositis, promoting wound healing, reducing scarring, reducing fibrosis, and treating auto-immune disease.
 21. A pharmaceutical composition comprising the nucleic acid molecule of claim 13, and one or more pharmaceutically acceptable excipients. 22.-30. (canceled)
 31. A protein molecule comprising a human Smad7 protein having amino acids 204-258 of the human Smad7 protein.
 32. (canceled)
 33. The protein molecule of claim 31, wherein the protein molecule has one or more biological activity selected from the group consisting of promoting healing in oral mucositis, promoting wound healing, reducing scarring, reducing fibrosis, and treating auto-immune disease.
 34. The protein molecule of claim 31, wherein the amino acid sequence further comprises a protein transduction domain.
 35. The protein molecule of claim 34, wherein the protein transduction domain is Tat. 36.-38. (canceled)
 39. A pharmaceutical composition comprising the protein molecule of claim 31, and one or more pharmaceutically acceptable excipients.
 40. A method for treating or preventing an inflammatory condition in a subject comprising providing to the subject a therapeutically effective amount of the pharmaceutical composition of claim
 21. 41. The method of claim 40, wherein the inflammatory condition is one or more of a chronic wound, skin inflammation, psoriasis, or an autoimmune disease.
 42. (canceled)
 43. A method for preventing or treating a disease or disorder in a subject comprising one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage in the subject comprising providing to the subject a therapeutically effective amount of the pharmaceutical composition of claim 21, wherein one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage is useful in preventing or treating the disease or disorder.
 44. The method of claim 43, wherein the disease or disorder includes one or more of chronic wounds, acute wounds, hypertrophic scarring, fibrosis, or oral mucositis. 45.-48. (canceled)
 49. The nucleic acid of claim 13, wherein the nucleotide sequence comprises nucleotides encoding amino acids 203-217 of the human Smad7 protein.
 50. The protein molecule of claim 31, wherein the Smad7 protein comprises amino acids 203-217 of the human Smad7 protein.
 51. A method for treating an inflammatory condition in a subject comprising providing to the subject a therapeutically effective amount of the protein molecule of claim
 50. 52. A method for preventing or treating a disease or disorder in a subject comprising one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage in the subject comprising providing to the subject a therapeutically effective amount of the protein of claim 50, wherein one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage is useful in preventing or treating the disease or disorder.
 53. A method for treating or preventing an inflammatory condition in a subject comprising providing to the subject a therapeutically effective amount of the pharmaceutical composition of claim
 39. 54. A method for preventing or treating a disease or disorder in a subject comprising one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage in the subject comprising providing to the subject a therapeutically effective amount of the pharmaceutical composition of claim 39, wherein one or more of increasing one or more of cell proliferation or cell migration, or preventing one or more of apoptosis or DNA damage is useful in preventing or treating the disease or disorder. 